Role of Actin DNase-I-Binding Loop in Myosin Subfragment 1-Induced Polymerization of G-actin: Implications for the Mechanism of Polymerization

AbstractProteolytic cleavage of actin between Gly42 and Val43 within its DNase-I-binding loop (D-loop) abolishes the ability of Ca-G-actin to spontaneously polymerize in the presence of KCl. Here we show that such modified actin is assembled into filaments, albeit at a lower rate than unmodified actin, by myosin subfragment 1 (S1) carrying the A1 essential light chain but not by S1(A2). S1 titration of pyrene-G-actin showed a diminished affinity of cleaved actin for S1, but this could be compensated for by using S1 in excess. The most significant effect of the cleavage, revealed by measuring the fluorescence of pyrene-actin and light-scattering intensities as a function of actin concentration at saturating concentrations of S1, is strong inhibition of association of G-actin-S1 complexes into oligomers. Measurements of the fluorescence of dansyl cadaverine attached to Gln41 indicate substantial inhibition of the initial association of G-actin-S1 into longitudinal dimers. The data provide experimental evidence for the critical role of D-loop conformation in both longitudinal and lateral, cross-strand actin-actin contact formation in the nucleation reaction. Electron microscopic analysis of the changes in filament-length distribution during polymerization of actin by S1(A1) and S1(A2) suggests that the mechanism of S1-induced polymerization is not substantially different from the nucleation-elongation scheme of spontaneous actin polymerization

The curious role of sarcomeric proteins in control of diverse processes in cardiac myocytes

Introduction Relatively recent developments in our understanding of sarcomeric proteins have expanded their role from the home of molecular motors generating force and shortening to a cellular organelle fully integrated in the control of structural, electrical, mechanical, chemical, and metabolic homeostasis. Even so, in some cases these diverse functions of sarcomeric proteins appear to remain a curiosity, not fully appreciated in the analysis of major controllers of cardiac function. This attitude toward the function of sarcomeric proteins in cardiac myocytes is summarized in the following definition of “curiosity,” which seems particularly apropos: “meddlesome; thrusting oneself into and taking an active part in others’ affairs.” We focus in this Perspective on how sarcomeric proteins function in integration with membrane channels and transporters in control of cardiac dynamics, especially in adrenergic control of cardiac function. Understanding these mechanisms at the level of cardiac sarcomeres took on special significance with the identification of mutations in sarcomeric proteins as the most common cause of familial hypertrophic and dilated cardiomyopathies. These mutations commonly lead to structural, electrical, and metabolic remodeling and to sudden death. These disorders indicate a critical role of processes at the level of the sarcomeres in homeostatic control of cardiac energetics, dynamics, and structure. Yet, control of Ca2+ delivery to and removal from the myofilaments has dominated discussions of mechanisms regulating cardiac contractility. We first provide an alternative perspective in which rate processes at the level of the sarcomeres appear to be dominant during the rise and maintenance of systolic elastance and of isovolumic relaxation. A discussion of established adrenergic mechanisms and newly understood anti-adrenergic mechanisms controlling sarcomere response to Ca2+ follows and expands on this perspective

Mechanical and kinetic effects of shortened tropomyosin reconstituted into myofibrils

The effects of tropomyosin on muscle mechanics and kinetics were examined in skeletal myofibrils using a novel method to remove tropomyosin (Tm) and troponin (Tn) and then replace these proteins with altered versions. Extraction employed a low ionic strength rigor solution, followed by sequential reconstitution at physiological ionic strength with Tm then Tn. SDS-PAGE analysis was consistent with full reconstitution, and fluorescence imaging after reconstitution using Oregon-green-labeled Tm indicated the expected localization. Myofibrils remained mechanically viable: maximum isometric forces of myofibrils after sTm/sTn reconstitution (control) were comparable (~84%) to the forces generated by non-reconstituted preparations, and the reconstitution minimally affected the rate of isometric activation (kact), calcium sensitivity (pCa50), and cooperativity (nH). Reconstitutions using various combinations of cardiac and skeletal Tm and Tn indicated that isoforms of both Tm and Tn influence calcium sensitivity of force development in opposite directions, but the isoforms do not otherwise alter cross-bridge kinetics. Myofibrils reconstituted with Δ23Tm, a deletion mutant lacking the second and third of Tm’s seven quasi-repeats, exhibited greatly depressed maximal force, moderately slower kact rates and reduced nH. Δ23Tm similarly decreased the cooperativity of calcium binding to the troponin regulatory sites of isolated thin filaments in solution. The mechanisms behind these effects of Δ23Tm also were investigated using Pi and ADP jumps. Pi and ADP kinetics were indistinguishable in Δ23Tm myofibrils compared to controls. The results suggest that the deleted region of tropomyosin is important for cooperative thin filament activation by calcium

Low Temperature Dynamic Mapping Reveals Unexpected Order and Disorder in Troponin*

Troponin is a pivotal regulatory protein that binds Ca2+ reversibly to act as the muscle contraction on-off switch. To understand troponin function, the dynamic behavior of the Ca2+-saturated cardiac troponin core domain was mapped in detail at 10 °C, using H/D exchange-mass spectrometry. The low temperature conditions of the present study greatly enhanced the dynamic map compared with previous work. Approximately 70% of assessable peptide bond hydrogens were protected from exchange sufficiently for dynamic measurement. This allowed the first characterization by this method of many regions of regulatory importance. Most of the TnI COOH terminus was protected from H/D exchange, implying an intrinsically folded structure. This region is critical to the troponin inhibitory function and has been implicated in thin filament activation. Other new findings include unprotected behavior, suggesting high mobility, for the residues linking the two domains of TnC, as well as for the inhibitory peptide residues preceding the TnI switch helix. These data indicate that, in solution, the regulatory subdomain of cardiac troponin is mobile relative to the remainder of troponin. Relatively dynamic properties were observed for the interacting TnI switch helix and TnC NH2-domain, contrasting with stable, highly protected properties for the interacting TnI helix 1 and TnC COOH-domain. Overall, exchange protection via protein folding was relatively weak or for a majority of peptide bond hydrogens. Several regions of TnT and TnI were unfolded even at low temperature, suggesting intrinsic disorder. Finally, change in temperature prominently altered local folding stability, suggesting that troponin is an unusually mobile protein under physiological conditions

Troponin Regulatory Function and Dynamics Revealed by H/D Exchange-Mass Spectrometry*

Muscle contraction is tightly regulated by Ca2+ binding to the thin filament protein troponin. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin using amide hydrogen exchange-mass spectrometry. Results were obtained in the presence of either saturation or non-saturation of the regulatory Ca2+ binding site in the NH2 domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D2O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D2O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca2+ binding altered dynamic properties (i.e. H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca2+-saturated TnC NH2 domain. More notably, Ca2+ also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH domain that contains the regulatory Ca2+ sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto the troponin highly extended structure suggests that cardiac troponin switches between alternative sets of intramolecular interactions, similar to previous intermediate resolution x-ray data of skeletal muscle troponin

Dual Regulatory Functions of the Thin Filament Revealed by Replacement of the Troponin I Inhibitory Peptide with a Linker*

Striated muscles are relaxed under low Ca2+ concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4–10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca2+ concentration conditions: inhibition of myosin subfragment 1 (S1)-thin filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to thin filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca2+ dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca2+ rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca2+ concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the thin filament regulatory Ca2+ sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca2+, and increases in myosin S1-thin filament ATPase rates in the presence of saturating Ca2+. The overall results indicate that cooperative myosin binding to Ca2+-free thin filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca2+ binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the thin filament