Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. III. Changes in the conformation of tropomyosin associated with functional states.

The local conformational changes in the tropomyosin molecule under various conditions were studied by means of fluorimetry using SH-directed fluorescent dyes, N-(1-anilinonaphthyl-4)maleimide (ANM) and N-(3-pyrene)maleimide (PRM). 1. The fluorescence intensity, polarization and the emmission maximum of ANM-tropomyosin were found to be susceptible to ionic strength, but in different ways. The changes in these parameters suggest that the fluorescence-labeled sulfhydryl group or groups become more buried in a hydrophobic internal region by salt-induced depolymerization of aggregate and by adding F-actin to tropomyosin. 2. Titration of the labeled tropomyosin with F-actin revealed a cooperative nature in ANM labeling and a simple saturation kinetics in PRM labeling. The dissociation constant of F-actin to PRM-tropomyosin was calculated to be 5.8-10(-6) M. 3. Temperature dependence of the fluorescence polarization showed a thermal transition in the conformation of ANM- or PRM-tropomyosin at around 30 degrees C. Flexibility or segmental motion of the region containing the fluorophore was suppressed significantly on adding troponin and markedly on adding F-actin. 4. Measurements of the quantum yield and polarization of the ANM-tropomyosin-F-actin complex suggested that troponin strengthened the binding between the two proteins and that Ca2+ reversed this effect.     

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. IV. Conformational changes in the region containing fluorescence-labeled sulfhydryl group(s) of troponin.

Conformational changes associated with the functional states of the molecule of troponin were studied using SH-direct fluorogenic reagents, N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM) and N-(1-anilinonaphthyl-4) maleimide (ANM). 1. The fluorescence parameters of ANM-troponin, intensity, and polarization, did not change on combining it with tropomyosin alone, but markedly changed when F-actin was further added to the system. 2. The conformation around the dye-labeled sulfhydryl group(s) was shown to be susceptible to Ca2+ in terms of fluorescence intensity of the label, thermal transition of the conformation, and the microenvironment near the label. 3. On addition of Ca2+, the fluorescence characteristics of the two systems, ANM-troponin . tropomyosin and ANM-troponin . tropomyosin . F-actin complexes, were altered in opposite directions. When BIPM was used in place of ANM, similar changes were observed: a simple decrease in the intensity when pCa was decreased from 7.4 to 5.5 in the system without F-actin and a sigmoidal increase in the range from pCa 7 to 6 in the system with F-actin. Heavy meromyosin, when added to the latter complex (the reconstituted thin filaments), made the profile of its Ca2+ concentration dependence of fluorescence similar to that of the former complex. When tropomyosin was labeled in place of troponin, similar results were obtained. The data obtained imply that the Ca2+-induced conformational changes of troponin are markedly modified when detached from actin, and that heavy meromyosin weakens the interaction of the troponin . tropomyosin complex with F-actin.     

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.     

Calcium ion accumulation and volume changes of isolated liver mitochondria. Reversal of calcium ion-induced swelling

1. The excessive accumulation of Ca²⁺ by mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing oxidizable substrate and phosphate led to extensive swelling and release of accumulated Ca²⁺ from the mitochondria. When the Ca²⁺ was removed from the medium by chelation with ethylene glycol bis(aminoethyl)tetra-acetate, the swelling was reversed in a respiration-dependent contraction. The contracted mitochondria were shown to have regained some degree of respiratory control. 2. The respiration-dependent contraction could be supported by electron transport through a restricted portion of the respiratory chain, and by substrates donating electrons at different levels in the respiratory chain. 3. Respiratory inhibitors appropriate to the substrate present completely inhibited the contraction. Uncoupling agents, and the inhibitors oligomycin and atractyloside, were without effect. 4. When the reversal of swelling had been prevented by respiratory inhibitors, the addition of ATP induced a contraction of the mitochondria. In the absence of added chelating agent the contraction was very slow. The ATP-induced contraction was completely inhibited by oligomycin and atractyloside, was incomplete in the presence of uncoupling agents and was unaffected by respiratory inhibitors. 5. The relationship between the energy requirements of respiration-dependent contraction and the requirements of ion transport and other contractile systems are discussed.     

In vitro modulation of filament bundling in f-actin and keratins by annexin II and calcium

Summary In our preliminary subcellular localization experiment we demonstrated that annexin II co-localized with submembranous actin in subpopulations of both cultured fibroblasts and keratinocytes. To investigate the physical interaction between annexin II and actin at the cell periphery, in vitro reconstitution experiments were carried out with keratins used as a control. Annexin II, isolated by immunoaffinity column chromatography, was found to exist as globular structures measuring 10 to 25 nm in diameter by rotary shadowing, similar to a previous report. We believe that these structures represent its polymeric forms. By negative staining, monomeric annexin II was detectable as tapered rods, measuring 6 nm in length and 1 to 2 nm in diameter. When annexin II was mixed with actin in 3 mM piperazine-N, N-bis-2-ethanesulfonic acid (PIPES) buffer with 10 mM NaCl₂, 2 mM MgCl₂ and 0.1 mM CaCl₂, thick twisting actin bundles formed, confirming previous reports. This bundling was much reduced when calcium was removed. In the presence of 5 mM ethylenediamine tetra-acetic acid (EDTA) in 5 mM tris, pH 7.2, keratins were found to form a network of filaments, which began to disassemble when the chelator was removed and became fragmented when 0.1 mM CaCl₂ was added. Keratins under the same conditions did not fragment when annexin II was present. These results suggest that annexin II, in conjunction with Ca²⁺, may be involved in a flexible system accommodating changes in the membrane cytoskeletal framework at the cell periphery in keratinocytes.     

Interactions of the inhibitory component of troponin, F-actin, and tropomyosin.

The interaction of the inhibitory component (TN I) of troponin and F-actin in the presence and absence of tropomyosin was studied by a number of physico-chemical techniques: i.e., gel filtration, ultracentrifugation, flow birefringence, viscosity and dynamic viscoelasticity measurements, and electron microscopy. The results indicated that TN I and F-actin interact with each other more strongly in the presence of tropomyosin than in its absence. The physiological implication of this finding is discussed.     

Cooperative regulation of myosin-actin interactions by a continuous flexible chain II: actin-tropomyosin-troponin and regulation by calcium

The model of myosin regulation by a continuous tropomyosin chain is generalized to a chain of tropomyosin-troponin units. Myosin binding to regulated actin is cooperative and initially inhibited by the chain as before. In the absence of calcium, myosin is further inhibited by the binding of troponin-I to actin, which through the whole of troponin pins the tropomyosin chain in a blocking position; myosin and TnI compete for actin and induce oppositely-directed chain kinks. The model predicts equilibrium binding curves for myosin-S1 and TnI as a function of their first-order affinities K(S1) and L(TI). Myosin is detached by the actin binding of TnI, but TnI is more efficiently detached by myosin when the kink size (typically nine to ten actin sites) spans the seven-site spacing between adjacent TnI molecules. An allosteric mechanism is used for coupling the detachment of TnI to calcium binding by TnC. With thermally activated TnI kinks (kink energy B approximately k(B)T), TnI also binds cooperatively to actin, producing cooperative detachment of myosin and biphasic myosin-calcium Hill plots, with Hill coefficients of 2 at high calcium and 4-6 at low calcium as observed in striated muscle. The theory also predicts the cooperative effects observed in the calcium loading of TnC.     

Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.

Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction.     

Effect of bivalent cations on the adenosine triphosphatase of actomyosin and its modification by tropomyosin and troponin

1. After removal of tropomyosin and troponin from the `natural'' actomyosin complex, the adenosine triphosphatase activity of the resulting `desensitized'' actomyosin is stimulated to the same extent by various bivalent cations with an ionic radius in the range 0·65–0·99å when tested at optimum concentration of the metal ion in the presence of 2·5mm-ATP at low ionic strength and pH7·6. Under identical conditions the adenosine triphosphatase activity of myosin alone is stimulated to an appreciable extent only by Ca²⁺ (ionic radius 0·99å). 2. Tropomyosin narrows the range of size of the stimulatory cations by inhibiting specifically the adenosine triphosphatase activity of `desensitized'' actomyosin when stimulated by Ca<sup>2+</sup> or the slightly smaller Cd<sup>2+</sup> (ionic radius 0·97å). Tropomyosin has no effect on the adenosine triphosphatase activity of `desensitized'' actomyosin when stimulated by the smaller cations, nor on the Ca²⁺-activated adenosine triphosphatase activity of myosin alone. 3. The adenosine triphosphatase activity of the `natural'' actomyosin system (containing tropomyosin and troponin) stimulated by the smallest cation, Mg²⁺ (ionic radius 0·65å), is low when the system is deprived of Ca²⁺ but high in the presence of small amounts of Ca²⁺. This sensitivity to Ca²⁺ seems to be a unique feature of the Mg²⁺-stimulated system. 4. The changes in specificity of the myosin adenosine triphosphatase activity in its requirement for bivalent cations caused by interaction with actin, tropomyosin and troponin primarily concern the size of the metal ions. The effects on enzymic properties of myofibrils due to tropomyosin and troponin can be demonstrated at low and at physiological ionic strength.     

Troponin-C-mediated calcium-sensitive changes in the conformation of troponin I detected by pyrene excimer fluorescence

Troponin I (TnI) from rabbit white skeletal muscle was labeled at cysteines 48 and 64 with the fluorescent reagent N-(1-pyrene)maleimide. The fluorescence spectra of pyrene-labeled TnI (pyr-TnI) exhibit peaks characteristic of pyrene in its monomeric form and an additional peak resulting from formation of excited dimers (excimers), indicating that the labeled cysteines are close together. Formation of a pyr-TnI-TnC complex in the absence of Ca2+ has little effect on the spectrum, but when Ca2+ is bound to the low-affinity sites of TnC there is a substantial decrease in excimer and a corresponding increase in monomer fluorescence. The involvement of the low-affinity sites in the Ca2+-induced effect is consistent with the fact that Mg2+ has no effect on pyrene fluorescence. On rapid mixing of the pyr-TnI-TnC complex with Ca2+ in a stopped-flow apparatus, most of the excimer decrease is complete within the instrumental dead time, indicating a rate constant k greater than 350 s-1, which is comparable to that of the conformational change in TnC resulting from Ca2+ binding to the low-affinity sites. Rapid mixing of the Mg2-TnC-pyr-TnI complex with Ca2+ yields similar results, suggesting that the type of metal ion present at the high-affinity sites has little, if any, effect on the probe. It has been suggested previously that Cys 48 and 64 are located in a TnT-binding region of TnI (Chong P.C.S. and Hodges, R.S. (1982) J. Biol. Chem. 255, 3757). Our results suggest that a Ca2+-induced structural change in the TnI-binding region of TnC could be transmitted to TnT by affecting the TnT-binding region of TnI as part of the chain of events in the regulation of muscle contraction.