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The Flexibility of Actin Filaments as Revealed by Fluorescence Resonance Energy Transfer

THE INFLUENCE OF DIVALENT CATIONS*
Open AccessPublished:May 07, 1999DOI:https://doi.org/10.1074/jbc.274.19.12996
      The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f′, was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility of the protein matrix within actin protomers (intramonomer flexibility). In both cases the relative increase as a function of temperature in f′ is larger in calcium-F-actin than in magnesium-F-actin in the range of 5–40 °C, which indicates that both the intramonomer and the intermonomer flexibility of the actin filaments are larger in calcium-F-actin than those in magnesium-F-actin. The intermonomer flexibility was proved to be larger than the intramonomer one in both the calcium-F-actin and the magnesium-F-actin. The distance between Gln41 and Cys374 residues was found to be cation-independent and did not change during polymerization at 21 °C. The steady-state fluorescence anisotropy data of fluorophores attached to the Gln41 or Cys374 residues suggest that the microenvironments around these regions are more rigid in the magnesium-loaded actin filament than in the calcium-loaded form.
      The tension generation in the striated muscle is performed through a series of chemical reactions by cyclic interaction of myosin with ATP and actin, and at least six intermediates are proposed for actomyosin ATPase in solution (
      • Stein L.A.
      • Schwarz R.P.
      • Chock P.B.
      • Eisenberg E.
      ,
      • Eisenberg E.
      • Greene L.E.
      ,
      • Brenner B.
      • Eisenberg E.
      ). On a cellular level in supramolecular complexes where stabilizing forces may modulate the hydrolysis process, some contribution from actin flexibility and dynamics to the contraction process cannot be excluded. This statement is supported by earlier and recent suggestions about the role of actin during the force development in muscle (
      • Wakabayashi K.
      • Sugimoto Y.
      • Tanaka H.
      • Ueno Y.
      • Takezawa Y.
      • Amemiya Y.
      ).
      Flexural rigidity experiments suggested that the actin filament was extensible (
      • Oosawa F.
      ). These findings were supported by electron microscopic measurements on the sarcomere in rigor fibers (
      • Suzuki S.
      • Sugi H.
      ). The extensibility of the thin filaments was also suggested by the changes of the spacings of the x-ray diffraction pattern during contraction (
      • Wakabayashi K.
      • Sugimoto Y.
      • Tanaka H.
      • Ueno Y.
      • Takezawa Y.
      • Amemiya Y.
      ,
      • Huxley H.E.
      • Stuart A.
      • Sosa H.
      • Irving T.
      ). Actin filaments were shown to be elastic and extensible by measuring the stiffness of the actin-tropomyosin complex with in vitronanomanipulation (
      • Kojima H.
      • Ishijima A.
      • Yanagida T.
      ). Polarization studies using fluorescent phalloidine on skinned rabbit psoas fibers have demonstrated that the generation of force was associated with a conformational change in the actin filament (
      • Prochniewicz-Nakayama C.
      • Yanagida T.
      • Oosawa F.
      ). The sliding of actin filaments is diminished by the cross-linking of actin subunits (
      • Prochniewicz E.
      • Yanagida T.
      ). Egelman et al. (
      • Egelman E.H.
      • Francis N.
      • DeRosier D.
      ) and later the workgroup in DeRosier's laboratory (
      • Stokes D.L.
      • DeRosier D.
      ) emphasized the existence of variations in the twist along the axes of isolated filaments, which increases the fluctuations of approximately 10° in the azimuthal angle between adjacent monomers. The fluctuations, bending and twisting motions, are modulated by myosin and actin-binding proteins (
      • Thomas D.D.
      • Seidel J.C.
      • Gergely J.
      ,
      • Galazkiewicz B.
      • Belágyi J.
      • Dabrowska R.
      ). The tighter binding of myosin to actin reduces the torsional motion of a small section of F-actin, as reported by standard transfer-EPR measurements (
      • Thomas D.D.
      • Seidel J.C.
      • Gergely J.
      ,
      • Mossakowska M.
      • Belágyi J.
      • Strzelecka-Golaszewska H.
      ). The change of the orientation of spin labels on F-actin during interaction with heavy meromyosin was also reported (
      • Prochniewicz E.
      • Yanagida T.
      ,
      • Yanagida T.
      • Nakase M.
      • Nishiyama K.
      • Oosawa F.
      ). It is also known that the actin monomers undergo conformational changes or slight rotation during contraction (
      • Yagi Y.
      • Matsubara I.
      ). The large free energy change caused by binding of the myosin head to actin is also able to generate conformational change in actin (
      • Geeves M.A.
      ).
      Experimental evidences suggest that the exchange of the bound cation can also modify the dynamic and conformational state of the actin filament. Cation-dependent changes in the mobility of the N-terminal segment (first 21 amino acids) of actin were observed performing nuclear magnetic resonance (NMR) experiments (
      • Heintz D.
      • Kany H.
      • Kalbitzer H.R.
      ). The torsional rigidity of actin filaments is sensitive to the nature of the bound cation, since this parameter is larger in calcium-F-actin than in magnesium-F-actin (
      • Yasuda R.
      • Miyata H.
      • Kinosita Jr., K.
      ). Orlova and Egelman (
      • Orlova A.
      • Egelman E.H.
      ) have shown that the bending flexibility of filaments polymerized from magnesium-actin is approximately four times larger than in the case of calcium-F-actin. Contrary to these data, other laboratories found no essential change in the filament flexibility using dynamic light scattering measurements (
      • Scharf R.E.
      • Newman J.
      ) or various other techniques to determine the persistence length of the filaments (
      • Isambert H.
      • Venier P.
      • Maggs A.C.
      • Fattoum A.
      • Kassab R.
      • Pantaloni D.
      • Carlier M.-F.
      ,
      • Steinmetz M.O.
      • Goldie K.N.
      • Aebi U.
      ). The direct measurement of the flexibility of single actin filaments corroborates this latter conclusion (
      • Yasuda R.
      • Miyata H.
      • Kinosita Jr., K.
      ). Using fluorescence methods Miki et al. (
      • Miki M.
      • Wahl P.
      • Auchet J.-C.
      ) observed that the binding of Ca2+ to actin increased the mobility of the fluorophore attached to Cys374. The results of our spectroscopic experiments indicated that filaments polymerized in the presence of Ca2+ were more flexible than the filaments of magnesium-actin (
      • Hild G.
      • Nyitrai M.
      • Belágyi J.
      • Somogyi B.
      ).
      The recently published actin powerstroke model was based on the length changes in actin filaments, which require conformational transitions in each monomer (
      • Schutt C.E.
      • Rozycki M.D.
      • Chick J.K.
      • Lindberg U.
      ). During the ATP hydrolysis cycle the myosin heads can adopt more than one conformation in interaction with actin, and the multiple modes of binding can relate to different actin conformations. Recently, the negative experimental results of the rotating cross-bridge model have led to suggestions of a more complex model of the muscle contraction. This model involves large scale conformational changes of myosin head in the light chain-binding domain that rotates relative to the actin-binding portion of the catalytic domain (
      • Huxley A.F.
      ,
      • Thomas D.D.
      • Ramachandran S.
      • Roopnarine O.
      • Hayden D.W.
      • Ostap M.E.
      ,
      • Cook R.
      ). The closure of the cleft on the actin-binding domains, which follows the release of the Pi, results in a specific interaction between the two proteins, and this interaction might be modulated by the actual dynamic and conformational states of both proteins.
      Although there are strong indications that actin is an active part of the contracting system, we are still far from understanding the details of the biological function of this abundant protein. The lack of complete understanding of the function of the actin in the contracting system can emphasize the importance of further investigations dealing with this matter.
      The principal aim of this study was to characterize the effect of divalent cations on the internal flexibility and the conformational states of actin filaments using the method of fluorescence resonance energy transfer. According to the fluorescence resonance energy transfer data presented in this paper, the calcium-F-actin is proved to be more flexible than the magnesium-F-actin in either the intermonomer or the intramonomer protein flexibility. The intermonomer flexibility is larger than the intramonomer one, regardless of the nature of the bound cation. In accordance with these flexibility data the steady-state anisotropy experiments indicate that the microenvironments of the Gln41 and Cys374 residues are more rigid in the Mg2+-saturated filaments than in calcium-F-actin.

      RESULTS AND DISCUSSION

      The actin monomer has one high-affinity and three or more lower-affinity (i.e. intermediate- and low-affinity) cation-binding sites (see Ref.
      • Estes J.E.
      • Selden L.A.
      • Kinosian H.J.
      • Gershman L.C.
      for review). It is very likely thatin vivo the high-affinity site is occupied by Mg2+, and the Mg2+ and K+ ions compete for the lower-affinity binding sites (
      • Estes J.E.
      • Selden L.A.
      • Kinosian H.J.
      • Gershman L.C.
      ). The ion composition of the buffer that was used in this study to prepare magnesium-F-actin can be considered as a reasonable model for the free ion concentrations of Mg2+ and K+ in the cytosol (
      • Estes J.E.
      • Selden L.A.
      • Kinosian H.J.
      • Gershman L.C.
      ). This preparation resulted in a magnesium-F-actin that contains Mg2+ at the high-affinity binding site and probably either Mg2+ or K+ at the lower-affinity sites. According to earlier publications the type of the cation at the lower-affinity binding sites might have an important biological effect (
      • Zimmerle C.T.
      • Patane K.
      • Frieden C.
      ). The calcium-actin filaments were polymerized in the presence of millimolar concentration (2 mm) of CaCl2. Following this procedure the Ca2+ in calcium-F-actin, similar to the Mg2+ in magnesium-F-actin samples, occupies the high-affinity binding site and probably competes with the K+ for the lower-affinity binding sites.
      In the present work we explored the differences between flexibilities of filaments polymerized from calcium-actin and magnesium-actin by investigating separately the intermonomer and the intramonomer flexibilities. To examine intermonomer flexibilities the donor IAEDANS and the acceptor IAF are attached to different actin protomers within the filament. The relatively low donor ratio in these samples (compared with that of actin without the donor) assures that there is no acceptor in the actin filament, which is in resonance transfer with two donor molecules (see Fig. 1B). Accordingly, in these experiments one is dealing with a single donor-multiple acceptor system (see “Materials and Methods”). In a different experimental setup, the double labeling of the actin monomer makes it possible to study intramonomer flexibility within the actin filament. In this case it was necessary to dilute the samples with unlabeled actin to exclude the possibility of interaction between donor and acceptor molecules located on neighboring protomers. Considering the atomic model of the actin filament (
      • Holmes K.C.
      • Popp D.
      • Gebhard W.
      • Kabsch W.
      ), it is very likely that the 10-fold dilution of the double labeled actin monomers with unlabeled monomers accurately separates the labeled monomers within the double helix of actin filaments (Fig. 1C). The experiments designed to monitor the reversibility of the temperature-induced changes in the fluorescence parameters gave evidence that the changes were reversible.
      The distance between the donor (IAEDANS at Cys374) and acceptor (FC at Gln41) molecules is 4.46 ± 0.07 nm and 4.49 ± 0.06 nm in the Ca2+- and Mg2+-loaded forms of the monomer, respectively, indicating that the exchange of the bound cation does not influence the relative position of the Gln41 and Cys374residues in the actin monomer. The data are in good accordance with the results of Moraczewska et al. (
      • Moraczewska J.
      • Strzelecka-Golaszewska H.
      • Moens P.D.J.
      • dos Remedios C.G.
      ), who found that the replacement of Ca2+ with Mg2+ produced no essential change in the distance between Gln41 and Cys374. These results are also in agreement with our recent observation that the distance between Lys61 and Cys374 of the actin monomer is cation-independent (
      • Nyitrai M.
      • Hild G.
      • Lakos Zs.
      • Somogyi B.
      ). The distance between Gln41 (Cα) and Cys374 (Sγ) residues is 4.1 nm according to the x-ray diffraction experiments (
      • Schutt C.E.
      • Myslik J.C.
      • Rozycki M.D.
      • Goonesekere N.C.W.
      • Lindberg U.
      ). The value of this parameter resolved in our experiments is somewhat longer. The relatively small difference between the x-ray and the fluorescence data might be due to the size of the applied fluorescent probes.
      The donor-acceptor distances (between residues Cys374 and Gln41) in the filament at room temperature are 4.45 ± 0.08 nm and 4.59 ± 0.09 nm in calcium-F-actin and magnesium-F-actin, respectively (TableI.), which indicates that the polymerization does not affect significantly the donor-acceptor distance. This is in agreement with Miki's conclusion (
      • Miki M.
      ) that the small domain in the actin monomer is substantially rigid and compact and only slightly sensitive to the binding of DNase I or myosin subfragment 1 or tropomyosin-troponin or polymerization. Above we speculate on the basis of the filament model (
      • Holmes K.C.
      • Popp D.
      • Gebhard W.
      • Kabsch W.
      ) that the 10-fold dilution of doubly labeled actin samples with unlabeled actin diminishes the intermonomer resonance energy transfer. The lack of the effect of polymerization on the donor-acceptor distance implies that this assumption was correct. The distance between the two labeled residues of the small domain was temperature-independent in magnesium-F-actin (Table I). This statement is apparently not true for the calcium-F-actin (Table I), since the value of the donor-acceptor distance shows a decreasing tendency with increasing temperature (for discussion, see below).
      Table IThe temperature dependence of the fluorescence quantum yield of the IAEDANS (ΦD), the Förster's critical distance of the IAEDANS-FC pair (Ro), the transfer efficiency measured in the intramonomer transfer experiments (E), and the calculated donor-acceptor distances (R) in calcium-F-actin and magnesium-F-actin
      TemperatureΦDR oER
      Ca2+Mg2+Ca2+Mg2+Ca2+Mg2+Ca2+Mg2+
      °Cnm%nm
      70.550.535.065.0166.863.14.504.58
      (±0.05)(±0.06)(±1.6)(±2.0)(±0.08)(±0.08)
      110.540.535.045.0066.362.74.504.58
      (±0.04)(±0.06)(±2.0)(±1.8)(±0.09)(±0.09)
      160.510.524.994.9966.162.14.474.59
      (±0.06)(±0.09)(±2.2)(±1.5)(±0.08)(±0.07)
      210.490.514.964.9765.761.64.454.59
      (±0.08)(±0.09)(±2.1)(±1.5)(±0.08)(±0.09)
      260.470.504.924.9665.660.84.424.61
      (±0.08)(±0.07)(±1.9)(±1.4)(±0.08)(±0.10)
      320.440.494.874.9365.260.04.384.61
      (±0.06)(±0.09)(±1.7)(±1.2)(±0.10)(±0.08)
      390.410.474.824.9164.558.74.364.63
      (±0.9)(±0.09)(±1.4)(±0.5)(±0.09)(±0.09)
      The S.E. of the mean are given in parentheses, except for the quantum yield where the error appears in the third digit.
      The cation dependence of the flexibility of the actin protomer within the filament can be characterized by measuring the temperature profile of the normalized transfer efficiency (Equations 6 and 8). In experiments dealing with intraprotomer interactions the temperature dependence of the relative f′ is proved to be substantially larger in calcium-F-actin than in magnesium-F-actin between 5 and 40 °C (Fig. 2A). The total change of 5% in the Mg2+-saturated form faces the 30% increase in the Ca2+-saturated form. The data set suggests that the protomer structure is more flexible in the Ca2+-loaded form of the actin filament than that in the magnesium-loaded form. The change in the relative f′ is very similar in calcium-F-actin to what was observed in the case of actin monomer by using a similar donor-acceptor pair (
      • Nyitrai M.
      • Hild G.
      • Lakos Zs.
      • Somogyi B.
      ). Accordingly, the flexibility of the small domain does not seem to be sensitive to polymerization in calcium-actin. Contrary to this, the relative change of f′ is smaller in magnesium-F-actin than that in magnesium-G-actin (
      • Nyitrai M.
      • Hild G.
      • Lakos Zs.
      • Somogyi B.
      ), indicating that in the Mg2+-loaded form this protein segment is more rigid in the filament than it is in the monomer.
      Figure thumbnail gr2
      Figure 2A, the cation dependence of the temperature profile of the relative f′ in F-actin resolved in the experiments dealing with intra-monomer flexibility. The donor was IAEDANS, and FC served as an acceptor. The actin concentration was 30–40 μm, while the labeled actin was present at 1–3 μm. B, the temperature profile of the relativef′ in calcium-F-actin and magnesium-F-actin resolved in the experiments dealing with intermonomer flexibility. The value of this parameter was calculated from the results of experiments with the IAEDANS-IAF donor-acceptor pair. The actin concentration was 30–40 μm.
      According to the results of intermonomer transfer experiments, the change of the relative f′ is larger in the calcium-F-actin than in the magnesium-F-actin (Fig. 2B), which suggests that the strength of the intermonomer interaction is stronger in the Mg2+-saturated filament. By comparing the data obtained in the experiments addressing intramonomer and intermonomer fluorescence energy transfer, one can conclude that the intramonomer flexibility is smaller than the intermonomer flexibility for both the calcium-F-actin and magnesium-F-actin (Fig. 2, A and B). Considering that in intermonomer energy transfer the contributions of the two kinds of acceptor populations (see also “Materials and Methods”) to the measured fluorescence energy transfer efficiency are probably similar (
      • Holmes K.C.
      • Popp D.
      • Gebhard W.
      • Kabsch W.
      ), in these experiments it is not possible to separate the flexural properties of the genetic helix and the two-started long-pitch helix. The increase in the amplitude of the relative fluctuation of the donor and acceptor molecules should result in an increase of the mean value of the energy transfer rate constant, <k ti> and therefore the measurable donor-acceptor distance, even if the equilibrium distance between the two labels remains unchanged (
      • Somogyi B.
      • Matkó J.
      • Papp S.
      • Hevessy J.
      • Welch G.R.
      • Damjanovich S.
      ). In the light of our present data regarding the cation-dependent flexural properties of the filament, it seems possible that the slight temperature dependence of the donor-acceptor distance measured in the calcium-F-actin is partly the result of a temperature-induced increase in the amplitude of the relative fluctuation of the donor and acceptor molecules.
      The interpretation of the results described above requires further spectral considerations. Both the temperature- and cation-induced changes in the shape of the emission spectra of the donor and the absorption spectra of the acceptor are negligible (data are not shown). Accordingly, the value of the overlap integral (Equation 5.) depends on neither the temperature nor the nature of the bound cation. Therefore it cannot contribute to the observed changes of f′ in the filaments. However, the value of the f′, and hence the relative f′, might depend on the orientation factor (κ2). Although this is the only parameter in the fluorescence energy transfer experiments which cannot be measured properly, the measurements of the steady-state anisotropy of both the donor and the acceptor molecules might provide information regarding the behavior of κ2. The anisotropy of IAEDANS and FC is cation-dependent in the actin filament (Fig. 3, A and B). The measured anisotropy values are larger in the Mg2+-saturated form than in the Ca2+-saturated one for both IAEDANS and FC, which can be taken as an indication of conformational differences between the calcium-F-actin and magnesium-F-actin. Interestingly, similar cation-induced change was not observed in the case of IAF (Fig. 3C). Taking into account that both IAEDANS and IAF are connected to the same amino acid (Cys374), the different cation sensitivity possibly originates from the application of different fluorophores. According to the results of Orlova and Egelman (
      • Orlova A.
      • Egelman E.H.
      ), there is a high-density bridge between the two strands of filament when the high-affinity cation-binding sites are occupied by Ca2+. This density bridge was not observed in magnesium-F-actin. They proposed that the presence of this bridge could be the result of the shift in the position of the C terminus. Therefore, the cation dependence of the fluorescence anisotropy in the case of IAEDANS and FC might reflect the cation-induced intramolecular rearrangement of the C-terminal segment within the actin filament. Although the exact nature of this rearrangement is not known, it seems to be possible that the formation of the density bridge in calcium-F-actin involves the modification of some of the connections between the C-terminal segment and the small domain of either the same or the neighboring protomers. The subdomain 1 (involving the Cys374 residue) is in close contact with the subdomain 2 (which contains the Gln41 residue) of the subsequent protomer within the long-pitch helix (
      • Holmes K.C.
      • Popp D.
      • Gebhard W.
      • Kabsch W.
      ). Accordingly, the formation of the high-density bridge in calcium-F-actin can result in a conformation where the microenvironments of the Cys374 and the Gln41 residues are more flexible than these microenvironments in the magnesium-F-actin.
      Figure thumbnail gr3
      Figure 3The temperature dependence of the steady-state fluorescence anisotropy of IAEDANS (A), FC (B), and IAF (C) in calcium-F-actin (filled circles) and magnesium-F-actin (open circles). The concentration of actin was 5 μm in these experiments (see “Materials and Methods”).
      The temperature sensitivity of the fluorescence anisotropy of all fluorophores is similar (Fig. 3, A–C), which suggests that the temperature-induced change in the value of the orientation factor is also similar in these cases. Accordingly, the change in the κ2 is probably not the source of the apparent cation-dependent variation in the value of the relativef′ in either the intermonomer or the intramonomer energy transfer experiments. All these data allow the conclusion that both the intramonomer and intermonomer flexibilities are larger in calcium-F-actin than in magnesium-F-actin. Furthermore, the results of the steady-state anisotropy measurements support the conclusion that the microenvironments of the Gln41 and Cys374 residues are more rigid in the magnesium-F-actin than in the calcium-F-actin.
      The bending and torsional flexibility of calcium-F-actin was found to be smaller (
      • Orlova A.
      • Egelman E.H.
      ) or similar (
      • Huxley A.F.
      ,
      • Thomas D.D.
      • Ramachandran S.
      • Roopnarine O.
      • Hayden D.W.
      • Ostap M.E.
      ,
      • Cook R.
      ,
      • Feuer G.
      • Molnár F.
      • Pettkó E.
      • Straub F.B.
      ) to that of magnesium-F-actin. However, we have shown here and in our previous work (
      • Hild G.
      • Nyitrai M.
      • Belágyi J.
      • Somogyi B.
      ) that the flexibility characteristic for intramolecular motions on a nanosecond time scale is larger in calcium-F-actin than in the Mg2+-saturated form of the filament. We have suggested (
      • Hild G.
      • Nyitrai M.
      • Belágyi J.
      • Somogyi B.
      ) that the apparent conflict could be resolved considering that the methods applied in our experiments and those used in the cited articles (
      • Orlova A.
      • Egelman E.H.
      ,
      • Huxley A.F.
      ,
      • Thomas D.D.
      • Ramachandran S.
      • Roopnarine O.
      • Hayden D.W.
      • Ostap M.E.
      ,
      • Cook R.
      ,
      • Feuer G.
      • Molnár F.
      • Pettkó E.
      • Straub F.B.
      ) provide information about intramolecular motions on a substantially different time scales.
      The structure of the magnesium-F-actin can be taken as a model of the thin filament in the relaxed state. The changes in the actin-associated layer lines in x-ray diffraction pattern during muscle activation (
      • Maeda Y.
      • Popp D.
      • McLaughlin S.M.
      ,
      • Wakabayashi K.
      • Saito H.
      • Moriwaki N.
      • Kobayashi T.
      • Tanaka H.
      ) and the differences of the layer lines observed between magnesium-F-actin and calcium-F-actin (
      • Orlova A.
      • Egelman E.H.
      ) are similar. Relying on these data Egelman and Orlova (
      • Egelman E.H.
      • Orlova A.
      ) proposed that the structure of the calcium-F-actin was corresponding to the thin filaments in the activated state. It is very likely that due to the slow exchange of the tightly bound divalent cation in actin the replacement of Mg2+ by Ca2+ does not occur under physiological conditions (
      • Estes J.E.
      • Selden L.A.
      • Kinosian H.J.
      • Gershman L.C.
      ). Accordingly, Egelman and Orlova (
      • Egelman E.H.
      • Orlova A.
      ) concluded that the activated state of the thin filament was probably induced by the binding of myosin. One might assume that the similarity of the structure of calcium-F-actin and the structure of the F-actin in the activated thin filaments can extend to intramolecular dynamic events occurring on a nanosecond timescale. Thus, considering that actin-myosin interaction can possibly utilize the strain energy stored in actin filaments (
      • Janmey P.A.
      • Hvidt S.
      • Oster G.F.
      • Lamb J.
      • Stossel T.P.
      • Hartvig J.H.
      ), the divalent cation-dependent changes in the intramolecular flexibility described in this study might be important in the efficient energy transduction of the muscle contraction.