Temperature dependence of the fluorescence intensity and anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to Cys374 of actin monomer was investigated to characterize conformational differences between Ca- and Mg-G-actin. The fluorescence lifetime is longer in Mg-G-actin than that in Ca-G-actin in the temperature range of 5-34 degrees C. The width of the lifetime distribution is smaller by 30% in Mg-saturated actin monomer at 5 degrees C, and the difference becomes negligible above 30 degrees C. 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.

The effect of Cr(VI) anion on an ergosterol-producing strain of eukaryotic yeast Candida albicans and its mutant with ergosterol-less membrane was studied with EPR spectroscopy. 5- and 14-doxyl stearic acid spin probes were used to label the protoplast membrane after removal of the cell wall. In control experiments, the mutant strain exhibited larger rigidity in the membrane than its parental strain. Addition of Cr(VI), at a minimum inhibitory concentration of 0.6 mM, increased the rotational mobility of the spin labels significantly and decreased the temperature of the structural changes in both strains, in the temperature range between 0 and 30 degrees C. The ergosterol-less mutant, having a membrane composition with increased polyunsaturated fatty acid content, exhibited higher Cr(VI) sensitivity. Treatment of the membrane with Cr(VI) for 10 min already resulted in an increase in membrane fluidity. An EPR signal of Cr(V) was detected which reached maximum amplitude after 120 min of treatment with Cr(VI). Further chemical reduction of Cr(V) in the absence of extracellular Cr(VI) led to a lack of detectable paramagnetic chromium intermediates within 200 min.

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 Ca2+ by Mg2+. The temperature dependence of the fluorescence lifetime and the anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) attached covalently to the Cys374 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 (Cys374). 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 Mg2+-loaded form of the actin filament between 6 degreesC and 28 degreesC, but this difference becomes negligible above 28 degreesC. The results of saturation transfer electron paramagnetic resonance measurements on maleimide spin-labeled actin filaments indicate that the replacement of Ca2+ by Mg2+ 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.

Our earlier fluorescence measurements using N-( I-pyreny1)iodoacetamide-labeled actin revealed that caldesmon interacts with G-actin accelerating its nucleation at low salt concentration and causing polymerization in the absence of salt [Galgzkiewicz, B., Mossakowska, M., Ositiska, H. & Dgbrowska, R. (1985) FEBS Lctt. 184, 144-1491, In this work the caldesmon-induced process of actin polymerization as well as the dynamic properties of the polymers formed have been investigated with the use of fluorescence, electron paramagnetic resonance (EPR) and electron microscopy techniques.Fluorescence titration of N-( 1 -pyrenyl)iodoacetamide-labeled actin with caldesmon showed saturation of the polymerization at a 1 : 3 molar ratio of caldesmoniactin monomer. Parallel pelleting experiments revealed, however, that the process of polymer formation is biphasic and only at higher concentrations of caldesmon does the copolymer contain around one caldesmonithree actin monomers. At low concentration of caldesmon a complex of one caldesmon/nine actin monomers is formed. EPR spectroscopy, using maleimide spin label bound at Cys374 of actin, also indicated that one caldesmon molecule polymerizes nine actin monomers. Taken together, these results might suggest the existence of weak and strong forms of actin binding to caldesmon and detection of only the latter by the fluorescence method.Copolymers of actin and caldesmon are indistinguishable from actin polymerized by salt with respect to their appearance in the electron microscope and their ability to interact with heavy meromyosin, although they are characterized by lower torsional flexibility as indicated by immobilization of spin labels attached to actin.Caldesmon is an actin-and calmodulin-binding protein present in relatively high quantities in smooth muscle [I, 21. Its in vitro binding to actin is accompanied by a decrease in the activity of actin-activated myosin ATPase [3 -61. Addition of calmodulin in the presence of C a Z f , relieves the inhibition of the ATPase activity as a result of its competition with actin for binding to caldesmon [3 -61. These features, along with its location on actin filaments [7], led to the speculation that caldesmon may play a role in the regulation of actin-myosin interaction in smooth muscle [8 -111. The mechanism of this regulation has not yet been clarified.Caldesmon has also been detected in all nonmuscle cells so far examined [12 -151 and it is believed that this protein is an important regulator of both motile activities and cell architecture [lo, 16, 171. The latter function can be fulfilled by caldesmon-mediated control of the actin assembly/disassembly process.It has been previously found by us [5, 171 and others [2, 8, 181 that caldesmon, in the absence of salt, interacts with Gactin to induce its polymerization and with F-actin to form a network or bundles of filaments. Both these processes, like the effect of caldesmon on actin-myosin interaction, are reversed by Caz+ and calmodulin.In this work we have combined fluorescence, e...

Rotational dynamics and ordering of myosin heads in glycerinated skeletal muscle fibres were studied using an isothiocyanate-based spin label attached to the fast-reacting thiol sites of myosin and were compared with data obtained for maleimide and iodoacetamide spin labels attached to the same sites. The ordering of probe molecules on the millisecond time scale in the rigor state, at sarcomere length 2.2-2.3 ? 0.1 pm, was static. Isothiocyanate probe molecules showed greater mobility; the segment holding the label rotated in the microsecond time range. In the saturation transfer EPR time domain, MgADP did not produce a significant change in the mobility of spin labels.The spectra of isothiocyanate spin-labelled fibres were analyzed in terms of two narrow distributions with mean angles of 75" and 56". In the rigor state, the fractions represented approximately 76% and 24% of the total EPR absorbance. In the presence of MgADP, the conventional EPR spectra showed large changes in the ordering of isothiocyanate probe molecules towards a new distribution, the population with a 6 ' value of 56% increased from 24% to 71% at the expense of the 75% population with no change in the mean angles of the distributions. In the case of maleimide and iodoacetamide spin-labelled fibres, however, the effect of MgADP on the probe angular distribution was small.Tension in muscle is generated by the interaction of myosin with actin, and muscle shortening is the result of thick and thin filaments sliding past one another. Time-resolved Xray diffraction has provided evidence that the changes of the reflection pattern are associated with the movement of crossbridges, and that the time course of the changes correlates with tension development [l]. A different approach for the study of molecular events during muscle contraction is provided by the use of motion-sensitive probe molecules. Paramagnetic and fluorescent labels attached to myosin or actin have recently been used to report molecular motions in protein complexes and muscle fibres [2-51. In glycerinated muscle fibres, paramagnetic probe molecules have a large orientation order with respect to the fibre axis, and in rigor they exhibit very slow motion in the saturation transfer EPR time domain [6, 71. Extended measurements by Thomas et al. [S] have shown that in the presence of ATP or even in rigor, but at a sarcomere length where the overlap of the two sets of filaments is reduced, the heads rotate freely with an effective correlation time in the microsecond time scale.In earlier investigations it was found that the mobility of the 4-iodoacetamido-2,2,6,6-tetramethylpiperidinooxyl (IASL)Correspondence to J. BelBgyi, Central Research Laboratory, University Medical School, H-7643 PCcs, Szigeti ut 12, HungaryAbbreviations. MSL, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl spin label; IASL, 4-iodoacetamido-2,2,6,6-tetramethylpiperidinooxyl spin label; TCSL, 4-isothiocyanato-2,2,6,6-tetramethylpiperidinooxyl spin label ; Nbs,, 5,5'-dithiobis(2-nitrobenzoic acid) ; ApsA, P',Ps-di(ade...