The Flexibility of Actin Filaments as Revealed by Fluorescence Resonance Energy Transfer. THE INFLUENCE OF DIVALENT CATIONS (original) (raw)
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Biophysical Journal, 1998
The principal aim of this investigation was to study the change of the protein flexibility and/or conformational properties of actin filaments upon the replacement of Ca 2ϩ by Mg 2ϩ . The temperature dependence of the fluorescence lifetime and the anisotropy decay of N-(iodoacetyl)-NЈ-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) attached covalently to the Cys 374 residue of actin were measured. Saturation transfer electron paramagnetic resonance (ST-EPR) experiments were also carried out using N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide (MSL) attached to the same residue (Cys 374 ). The Arrhenius analysis of the temperature dependence of the fluorescence lifetimes shows that for Mg-F-actin, both the activation energy (E*) and the frequency factor (A) are smaller than they are for Ca-F-actin. The longer rotational correlation times resolved in the fluorescence experiments are larger in the Mg 2ϩ -loaded form of the actin filament between 6°C and 28°C, but this difference becomes negligible above 28°C. The results of saturation transfer electron paramagnetic resonance measurements on maleimide spin-labeled actin filaments indicate that the replacement of Ca 2ϩ by Mg 2ϩ induced a decrease of the mobility of the label on the sub-millisecond time scale. Based upon these results, we concluded that the filaments polymerized from Ca-actin are more flexible than the filaments of Mg-actin.
Biophysical Journal, 1998
A fluorescence resonance energy transfer (FRET) parameter, fЈ (defined as the average transfer efficiency, ͗E͘, normalized by the actual fluorescence intensity of the donor in the presence of acceptor, F DA ), was previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions. The temperature profile of this parameter was used to detect the change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca 2ϩ by Mg 2ϩ . The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-NЈ-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The fЈ increases with increasing temperature over the whole temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26°C, whereas an opposite tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26°C that was not detected in the case of Mg-G-actin. In the temperature range between 6°C and 26°C the slope of the temperature profile of fЈ is the same for Ca-G-actin and Mg-G-actin, suggesting that the flexibility of the protein matrix between the two labels is identical in the two forms of actin.
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.
Biophysical Journal, 1997
Temperature dependence of the fluorescence intensity and anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1naphthyl)ethylenediamine attached to Cys374 of actin monomer was investigated to characterize conformational differences between Caand Mg-G-actin. The fluorescence lifetime is longer in Mg-G-actin than that in Ca-G-actin in the temperature range of 5-34oC. The width of the lifetime distribution is smaller by 30% in Mg-saturated actin monomer at 50C, and the difference becomes negligible above 300C. The semiangle of the cone within which the fluorophore can rotate is larger in Ca-G-actin at all temperatures. Electron paramagnetic resonance measurements on maleimide spin-labeled (on Cys374) monomer actin gave evidence that exchange of Ca2+ for Mg2+ induced a rapid decrease in the mobility of the label immediately after the addition of Mg2+. These results suggest that the C-terminal region of the monomer becomes more rigid as a result of the replacement of Ca2+ by Mg2+. The change can be related to the difference between the polymerization abilities of the two forms of G-actin.
Models of the actin monomer and filament from fluorescence resonance-energy transfer
European Journal of Biochemistry, 1992
We have developed algorithms for combining fluorescence resonance-energy transfer (FRET) efficiency measurements into structural models which predict the relative positions of the chemical groups used in FRET. We used these algorithms to construct models of the actin monomer and filament derived solely from FRET measurements based on seven distinct loci. We found a mirrorimage pair of monomer models which best fit the FRET data. One of these models agrees well with the atomic-resolution crystal structure recently published by Kabsch et al. ]. The rootmean-square deviation between this FRET model and the crystal structure was about 0.9 nm. Other macromolecular models assembled from FRET measurements are likely to have a similar resolution. The largest discrepancy was for the CyslO locus which deviated 1.44 nm from the crystal position. We discuss the limitations of the FRET method that may have contributed to this discrepancy, and conclude that the CyslO FRET data have probably located CyslO incorrectly in the FRET monomer model. Using the FRET monomer models, we found three orientations in the filament which best fit the intermonomer FRET data. These orientations differ substantially from the atomic-resolution filament model proposed by the ], largely because of the discrepancies in the CyslO data. These data should probably be excluded from the analysis; however, this would leave too few measurements to assemble a filament model. In the near future, we hope to obtain additional FRET measurements to other actin loci so that the filament modelling can be done without the CyslO data.
Biophysical Journal, 1999
Conformational changes in subdomain 2 of actin were investigated using fluorescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) covalently attached to Gln 41 . Examination of changes in the fluorescence emission spectra as a function of time during Ca 2ϩ /Mg 2ϩ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin confirmed a profound influence of the type of nucleotide but failed to detect a significant cationdependent difference in the environment of Gln 41 . No significant difference between Ca-and Mg-actin was also seen in the magnitude of the fluorescence changes resulting from the polymerization of these two actin forms. Evidence is presented that earlier reported cation-dependent differences in the conformation of the loop 38 -52 may be related to time-dependent changes in the conformation of subdomain 2 in DED-or DC-labeled G-actin, accelerated by substitution of Mg 2ϩ for Ca 2ϩ in CaATP-G-actin and, in particular, by conversion of MgATP-into MgADP-G-actin. These spontaneous changes are associated with a denaturation-driven release of the bound nucleotide that is promoted by two effects of DED or DC labeling: lowered affinity of actin for nucleotide and acceleration of ATP hydrolysis on MgATP-G-actin that converts it into a less stable MgADP form. Evidence is presented that the changes in the environment of Gln 41 accompanying actin polymerization result in part from the release of P i after the hydrolysis of ATP on the polymer. A similarity of this change to that accompanying replacement of the bound ATP with ADP in G-actin is discussed.
PubMed, 1981
A fluorescent reagent, N-(1-pyrenyl)iodoacetamide, was conjugated to rabbit skeletal muscle actin at the site of the most reactive sulfhydryl group, and fluorescence characteristics (excitation and emission spectra, quantum yields, lifetimes) of the conjugate were investigated. Associated with polymerization of labelled G-actin, the fluorescence intensity at 407 nm, after excitation at 365 nm, was enhanced by a factor of about 25. It was reduced to about 25% on the binding of heavy meromyosin (or subfragment 1). The results suggest that binding of heavy meromyosin to the protomer of F-actin alters the local structure of the protomer towards a G-actin-like one.
Biophysical Journal, 1995
Gln-41 on G-actin was specifically labeled with a fluorescent probe, dansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2+ with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2' quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCI2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.
New Aspects of the Spontaneous Polymerization of Actin in the Presence of Salts
Journal of Molecular Biology, 2009
The mechanism of salt-induced actin polymerization involves the energetically unfavorable nucleation step, followed by filament elongation by the addition of monomers. The use of a bifunctional cross-linker, N,N′-(1,4phenylene)dimaleimide, revealed rapid formation of the so-called lower dimers (LD) in which actin monomers are arranged in an antiparallel fashion. The filament elongation phase is characterized by a gradual LD decay and an increase in the yield of "upper dimers" (UD) characteristic of F-actin. Here we have used 90°light scattering, electron microscopy, and N, N′-(1,4-phenylene)dimaleimide cross-linking to reinvestigate relationships between changes in filament morphology, LD decay, and increase in the yield of UD during filament growth in a wide range of conditions influencing the rate of the nucleation reaction. The results show irregularity and instability of filaments at early stages of polymerization under all conditions used, and suggest that an earlier documented coassembling of LD with monomeric actin contributes to the initial disordering of the filaments rather than to the nucleation of polymerization. The effects of the type of G-actin-bound divalent cation (Ca 2+ /Mg 2+ ), nucleotide (ATP/ ADP), and polymerizing salt on the relation between changes in filament morphology and progress in G-actin-to-F-actin transformation show that ligand-dependent alterations in G-actin conformation determine not only the nucleation rate but also the kinetics of ordering of the filament structure in the elongation phase. The time courses of changes in the yield of UD suggest that filament maturation involves cooperative propagation of "proper" interprotomer contacts. Acceleration of this process by the initially bound MgATP supports the view that the filament-destabilizing conformational changes triggered by ATP hydrolysis and P i liberation during polymerization are constrained by the intermolecular contacts established between MgATP monomers prior to ATP hydrolysis. An important role of contacts involving the DNase-I-binding loop and the C-terminus of actin is proposed.
Biophysical Journal, 1984
Fluorescence photobleaching recovery (FPR) was measured to determine the diffusion coefficient of fluorescein-labeled G-actin in low-salt buffer. The result obtained, 7.15 ± 0.35 x 10-7 Cm2/S, is in good agreement with that computed from the molecular weight, partial specific volume, and sedimentation coefficient, but is higher than previously obtained values. It is demonstrated from theory that at low ionic strength, the electrostatic contribution to the intrinsic viscosity leads to an overestimate of the hydrodynamic eccentricity of G-actin. Data from FPR, sedimentation, and fluorescence polarization experiments all indicate that the true low-salt form of the actin monomer has an axial ratio _3.0. The G-F transformation of actin was also observed by measurement of FPR during the assembly phase, in the steady state, and in the presence of ligands such as cytochalasin and aldolase. Each FPR record in general yields three data: relative proportion of rapidly and slowly diffusing actin, diffusion coefficient for the high-mobility fraction, and a mean diffusion coefficient for the low-mobility fraction. A relation between the mean low-mobility diffusion coefficient and the number-average filament length is derived and applied to the analysis of FPR data. Under typical conditions, the average filament length was >> 10 ,um in the steady state. Cytochalasin D was found to decrease filament length and total amount of filament proportionally; total filament number was not greatly affected. In all polymerizations of G-actin, the high-mobility material observed in situ was found to be essentially monomeric actin.