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Calcium binding to troponin C (TnC) activates striated muscle contraction by removing TnI (troponin I) from its inhibitory site on actin. Troponin T (TnT) links TnI with tropomyosin, causing tropomyosin to move from an inhibitory position on actin to an activating position. Positive charges within the C-terminal region of human cardiac TnT limit Ca2+ activation. We now show that the positively charged region of TnT has an even larger impact on skeletal muscle regulation. We prepared one variant of human skeletal TnT that had the C-terminal 16 residues truncated (Δ16) and another with an added C-terminal Cys residue and Ala substituted for the last 6 basic residues (251C-HAHA). Both mutants reduced (based on S1 binding kinetics) or eliminated (based on acrylodan-tropomyosin fluorescence) the first inactive state of actin at

Calcium binding to troponin C (TnC) is insufficient for full activation of myosin ATPase activity by actin-tropomyosin-troponin. Previous attempts to investigate full activation utilized ATP-free myosin or chemically modified myosin to stabilize the active state of regulated actin. We utilized the Δ14-TnT and the A8V-TnC mutants to stabilize the activated state at saturating Ca(2+) and to eliminate one of the inactive states at low Ca(2+). The observed effects differed in solution studies and in the more ordered in vitro motility assay and in skinned cardiac muscle preparations. At saturating Ca(2+), full activation with Δ14-TnT·A8V-TnC decreased the apparent K(M) for actin-activated ATPase activity compared to bare actin filaments. Rates of in vitro motility increased at both high and low Ca(2+) with Δ14-TnT; the maximum shortening speed at high Ca(2+) increased 1.8-fold. Cardiac muscle preparations exhibited increased Ca(2+) sensitivity and large increases in resting force with either Δ14-TnT or Δ14-TnT·A8V-TnC. We also observed a significant increase in the maximal rate of tension redevelopment. The results of full activation with Ca(2+) and Δ14-TnT·A8V-TnC confirmed and extended several earlier observations using other means of reaching full activation. Furthermore, at low Ca(2+), elimination of the first inactive state led to partial activation. This work also confirms, in three distinct experimental systems, that troponin is able to stabilize the active state of actin-tropomyosin-troponin without the need for high-affinity myosin binding. The results are relevant to the reason for two inactive states and for the role of force producing myosin in regulation.

Striated muscle is activated by myosin- and actin-linked processes, with the latter being regulated through changes in the position of tropomyosin relative to the actin surface. The C-terminal region of cardiac troponin T (TnT), a tropomyosin-associated protein, is required for full TnT inactivation at low Ca(2+) and for limiting its activation at saturating Ca(2+). Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca(2+)-dependent conformational changes, and whether these residues affect cardiac muscle contraction. We generated a human cardiac TnT variant in which we replaced seven C-terminal Lys and Arg residues with Ala and added a Cys residue at either position 289 or 275 to affix a fluorescent probe. At pCa 3.7, actin filaments containing high-alanine TnT had an elevated ATPase rate like that obtained when the last TnT 14 residues were deleted. Acrylodan-tropomyosin fluorescence changes and S1-actin binding kinetics revealed that at pCa 8, the high-alanine TnT-containing filaments did not enter the first inactive state. FRET analyses indicated that the C-terminal TnT region approached Cys-190 of tropomyosin as actin filaments transitioned to the inactive B state; that transition was abolished with high-alanine TnT. High-alanine TnT-containing cardiac muscle preparations had increased Ca(2+) sensitivity of both steady-state isometric force and sinusoidal stiffness as well as increased maximum steady-state isometric force and sinusoidal stiffness. We conclude that C-terminal basic residues in cardiac TnT are critical for the regulation of cardiac muscle contraction.

Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, ‘shutdown’ state with the filament-forming tail folded onto the two heads1, which prevents filament formation and inactivates the motors2. The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 A in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations. The structure of myosin-2 in the shutdown state reveals how the shutdown state is stabilized and how phosphorylation of light chains allows myosin to be activated.

It has been shown that not only calcium but also strong binding myosin heads contribute to thin filament activation in isometrically contracting animal fast-twitch and cardiac muscle preparations. This behavior has not been studied in human muscle fibers or animal slow-twitch fibers. Human slow-twitch fibers are interesting since they contain the same myosin heavy chain isoform as the human heart. To explore myosin-induced activation of the thin filament in isometrically contracting human slow-twitch fibers, the endogenous troponin complex was exchanged for a well-characterized fast-twitch skeletal troponin complex labeled with the fluorescent dye N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (fsTn-IANBD). The exchange was ≈70% complete (n = 8). The relative contributions of calcium and strong binding cross-bridges to thin filament activation were dissected by increasing the concentration of calcium from relaxing (pCa 7.5) to saturating levels (pCa 4.5) before and after incubating the exchanged fibers in the myosin inhibitor para-aminoblebbistatin (AmBleb). At pCa 4.5, the relative contributions of calcium and strong binding cross-bridges to thin filament activation were ≈69 and ≈31%, respectively. Additionally, switching from isometric to isotonic contraction at pCa 4.5 revealed that strong binding cross-bridges contributed ≈29% to thin filament activation (i.e., virtually the same magnitude obtained with AmBleb). Thus, we showed through two different approaches that lowering the number of strong binding cross-bridges, at saturating calcium, significantly reduced the activation of the thin filament in human slow-twitch fibers. The contribution of myosin to activation resembled that which was previously reported in rat cardiac and rabbit fast-twitch muscle preparations. This method could be applied to slow-twitch human fibers obtained from the soleus muscle of cardiomyopathy patients. Such studies could lead to a better understanding of the effect of point mutations of the cardiac myosin head on the regulation of muscle contraction and could lead to better management by pharmacological approaches.

Activation of striated muscle contraction occurs in response to Ca(2+) binding to troponin C. The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin-catalyzed ATP hydrolysis. It now appears that the C-terminal 14 amino acids of cardiac troponin T (TnT) control the level of activity at both low and high Ca(2+). We made a series of C-terminal truncation mutants of human cardiac troponin T, isoform 2, to determine if the same residues of TnT are involved in the low and high Ca(2+) effects. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca(2+). Changes in acrylodan tropomyosin fluorescence and the degree of Ca(2+) stimulation of the rate of binding of rigor myosin subfragment 1 to pyrene-labeled actin-tropomyosin-troponin were measured at low Ca(2+). These measurements define the distribution of actin-tropomyosin-troponin among the three regulatory states. Residues SKTR and GRWK of TnT were required for the functioning of TnT at both low and high Ca(2+). Thus, the effects on forming the inactive B-state and in retarding formation of the active M-state require the same regions of TnT. We also observed that the rate of binding of rigor subfragment 1 to pyrene-labeled regulated actin at saturating Ca(2+) was higher for the truncation mutants than for wild-type TnT. This violated an assumption necessary for determining the B-state population by this kinetic method.

Activation of striated muscle contraction occurs in response to Ca2+binding to troponin C (TnC). The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin catalyzed ATP hydrolysis. It now appears that the levels of activity at both low and saturating Ca2+are modulated by the C-terminal 14 amino acids of cardiac troponin T (TnT). We made a series of mutants of human cardiac troponin T, isoform 2, with deletions from the C-terminal end: Δ4, Δ6, Δ8, Δ10 and Δ14. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca2+, the change in acrylodan tropomyosin fluorescence at low Ca2+, and the degree of Ca2+stimulation of the rate of binding of rigor myosin S1 to pyrene-labeled actin-tropomyosin-troponin. Together, these measurements define the distribution of actin-tropomyosin-troponin among the 3 regulatory states. Results from rates of rigor S1 binding deviated from other measurements when > 8 residues of TnT were deleted. That deviation was due to increased rates of binding of rigor S1 to pyrene-labeled actin with truncated TnT at saturating Ca2+. Such behavior violated a key assumption in the determination of the B state by this method. Nevertheless, all methods show that as residues were removed from the C-terminus of TnT there was approximately a proportional loss of the inactive B state at low Ca2+and an increase in the active M state at saturating Ca2+. Most of the C-terminal 14 residues of human cardiac troponin T are essential for forming the inactive B state at low Ca2+and for limiting the formation of the active M state at saturating Ca2+.

Striated muscle contraction is regulated by the actin-associated proteins tropomyosin and troponin. The extent of activation of myosin ATPase activity is lowest in the absence of both Ca(2+) and activating cross-bridges (i.e., S1-ADP or rigor S1). Binding of activating species of myosin to actin at a saturating Ca(2+) concentration stabilizes the most active state (M state) of the actin–tropomyosin–troponin complex (regulated actin). Ca(2+) binding alone produces partial stabilization of the active state. The extent of stabilization at a saturating Ca(2+) concentration depends on the isoform of the troponin subunits, the phosphorylation state of troponin, and, in the case of cardiac muscle, the presence of hypertrophic cardiomyopathy-producing mutants of troponin T and troponin I. Cardiac dysfunction is also associated with mutations of troponin C (TnC). Troponin C mutants A8V, C84Y, and D145E increase the Ca(2+) sensitivity of ATPase activity. We show that these mutants change the distribution of regulated actin states. The A8V and C84Y TnC mutants decreased the inactive B state distribution slightly at low Ca(2+) concentrations, but the D145E mutants had no effect on that state. All TnC mutants increased the level of the active M state compared to that of the wild type, at a saturating Ca(2+) concentration. Troponin complexes that contained two mutations that stabilize the active M state, A8V TnC and Δ14 TnT, appeared to be completely in the active state in the presence of only Ca(2+). Because Ca(2+) gives full activation, in this situation, troponin must be capable of positioning tropomyosin in the active M state without the need for rigor myosin binding.

Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP, although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins that stabilize myosin filaments and that stabilize actin-myosin interactions have been identified. Fesselin or synaptopodin 2 appears to be another such protein. Rapid kinetic measurements and electron microscopy demonstrated that fesselin, isolated from turkey gizzard muscle, reduced the rate of dissociation of myosin filaments. Addition of fesselin increased both the length and thickness of myosin filaments. The rate of detachment of myosin, but not heavy meromyosin, from actin was also greatly reduced by fesselin. Data from this study suggest that fesselin stabilizes myosin filaments and tethers myosin to actin. These results support the view that one role of fesselin is to organize contractile units of myosin and actin.

Fesselin or avian synaptopodin 2 is a member of the synaptopodin family of actin binding proteins. Fesselin promotes G-actin polymerization and the formation of large actin complexes that can be collected by low-speed centrifugation. Because of the potential role of fesselin in some cancers and its effects on actin, we further investigated the effect of fesselin on actin. Fesselin initiated actin polymerization under a variety of conditions, including the virtual absence of salt. Actin filaments formed at low salt concentrations in the presence of fesselin were similar to filaments polymerized in the presence of 100 mM KCl. In both cases, the filaments were long and straight with a common orientation. Highly ordered actin bundles formed with increasing times of incubation. Blockers of actin growth at the barbed end (cytochalasin D and CapZ) did not prevent fesselin from polymerizing actin. Low concentrations of fesselin increased the critical concentration of actin. Both observations are consistent with preferential growth at the pointed end of actin filaments. These results indicate a role of fesselin in organizing cellular actin. These and other results indicate that fesselin is part of a cellular actin organizing center.

The R146G mutation of troponin I (TnI) is associated with hypertrophic cardiomyopathy in humans. Earlier data pointed to stabilization of the intermediate, C state, of actin-tropomyosin-troponin by this mutant. Because cardiac disorders appear to be linked to changes in regulated actin distributions, we determined the extent to which the R146G TnI mutant alters the distribution of states at low and high Ca(2+) concentrations. We show, from measurements of the kcat for actin-activated ATPase activity at saturating Ca(2+) concentrations, that R146G TnI reduced the population of the active, M, state to 25% of the wild-type level. Together with acrylodan-tropomyosin fluorescence measurements of the B state, it appeared that the C state was populated at ∼91% of the total for the R146G TnI-containing actin filaments. The C state was also more heavily populated at low Ca(2+) concentrations. Acrylodan-tropomyosin fluorescence changes showed a large diminution in the inactive state value relative to the wild-type value without a comparable increase in the active state. Furthermore, the rate of binding of rigor S1 to pyrene-labeled actin filaments containing R146G TnI was faster than the rate of binding to wild-type filaments at low free Ca(2+) concentrations. These results indicate that the inhibitory region of TnI affects the B-C and M-C equilibria of actin-tropomyosin-troponin. The observation that a mutation in the inhibitory region affects the M-C equilibrium may point to a novel regulatory interaction.

Loss of strength in human and animal models of aging can be partially attributed to a well-recognized decrease in muscle mass; however, starting at middle-age, the normalized force (force/muscle cross-sectional area) in the knee extensors and single muscle fibers declines in a curvilinear manner. Strength is lost faster than muscle mass and is a more consistent risk factor for disability and death. Reduced expression of the voltage sensor Ca2+ channel α1 subunit (Cav1.1) with aging leads to excitation-contraction uncoupling, which accounts for a significant fraction of the decrease in skeletal muscle function. We recently reported that in addition to its classical cytoplasmic location, fast skeletal muscle troponin T3 (TnT3) is fragmented in aging mice, and both full-length TnT3 (FL-TnT3) and its carboxyl-terminal (CT-TnT3) fragment shuttle to the nucleus. Here, we demonstrate that it regulates transcription of Cacna1s, the gene encoding Cav1.1. Knocking down TnT3 in vivo downregulated Cav1.1. TnT3 downregulation or overexpression decreased or increased, respectively, Cacna1s promoter activity, and the effect was ablated by truncating the TnT3 nuclear localization sequence. Further, we mapped the Cacna1s promoter region and established the consensus sequence for TnT3 binding to Cacna1s promoter. Systemic administration of BDA-410, a specific calpain inhibitor, prevented TnT3 fragmentation, and Cacna1s and Cav1.1 downregulation and improved muscle force generation in sedentary old mice., Instituto de Investigaciones Bioquímicas de La Plata

Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca(2+) binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin-tropomyosin-actin complex. Herein, we report a calorimetric study of Ca(2+) binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca(2+) binding to both the high-affinity and the low-affinity domain of HcTnC. Ca(2+) binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca(2+) binding to the regulatory domain. This pattern stems from changes that occur at the Ca(2+) site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC(1-89) support this claim. The values of the Gibbs free energy for Ca(2+) binding to the N-domain in the full-length protein and to the isolated domain (HcTnC(1-89)) are similar; however, differences in the entropic and enthalpic contributions to the free energy provide supporting evidence for the cooperativity of the C-domain and the N-domain. Thermograms obtained at two additional temperatures (10 and 37 °C) revealed interesting trends in the enthalpies and entropies of binding for both thermodynamic events. This allowed the determination of the change in heat capacity (∆C(p)) from a plot of ∆H verses temperature and may provide evidence for positive cooperativity of Ca(2+) binding to the C-domain.

Striated muscle contraction is regulated primarily through the action of tropomyosin and troponin that are bound to actin. Activation requires Ca(2+) binding to troponin and/or binding of high affinity myosin complexes to actin. Mutations within components of the regulatory complex may lead to familial cardiomyopathies and myopathies. In several cases examined, either physiological or pathological changes in troponin alter the distribution among states of actin-tropomyosin-troponin that differ in their abilities to stimulate myosin ATPase activity. These observations open possibilities for managing disorders of the troponin complex. Furthermore, analyses of mutant forms of troponin give insights into the regulation of striated muscle contraction.

In this special edition of the Journal of Muscle Research and Cell Motility, we recall the lives and scientific contributions of Michael and Kate Bárány, who died in 2011. Michael and Kate were Holocaust survivors who went on to become leading researchers in muscle contraction. Their research topics included myosin isoforms, phosphorylation as a regulator of muscle contraction and the application of NMR to study muscle metabolism. They were deeply committed to science and to fostering the careers of young investigators.

Changes in the muscle regulatory protein complex, troponin, are important for modulation of activity and may occur as a result of disease-causing mutations. Both increases and decreases in the rate of ATP hydrolysis by myosin may occur as dictated by changes in the distribution of actin-tropomyosin-troponin among its different states. It is important to measure the rates of transition among these states to study physiological adaptation and disease processes. We show here that acrylodan or pyrene probes on tropomyosin can be used to monitor the transition from active to intermediate and inactive states of actin-tropomyosin-troponin. Transitions measured in the absence of calcium had two phases, as previously reported for some other probes on troponin and actin. The first step was a rapid equilibrium that favored the formation of the intermediate state and had an apparent rate constant less than that of S1-ATP dissociation. The second fluorescence transition was slower, with an apparent constant that increased from approximately 5 to 80/s over a range of 1-37 degrees C. Only the initial rapid transition was seen in the presence of saturating calcium. The acrylodan probe had the advantage of yielding a larger signal than the pyrene probe. Furthermore, the acrylodan signal decreased in going from the active state to the intermediate state, and then increased upon going to the inactive state.

We examined four cardiomyopathy-causing mutations of troponin I that appear to disturb function by altering the distribution of thin filament states. The R193H (mouse) troponin I mutant had greater than normal actin-activated myosin-S1 ATPase activity in both the presence and absence of calcium. The rate of ATPase activity was the same as that of the wild-type at near-saturating concentrations of the activator, N-ethylmaleimide-S1. This mutant appeared to function by stabilizing the active state of thin filaments. Mutations D191H, R146G, and R146W had lower ATPase activities in the presence of calcium, but higher activities in the absence of calcium. These effects were most pronounced with mutations at position 146. For all three mutants the rates were similar to those of the wild-type at near-saturating concentrations of N-ethylmaleimide-S1. These results, combined with previous results, show that any alteration in the normal distribution of actomyosin states is capable of producing cardiomyopathy. The results of the D191H, R146G, and R146W mutations are most readily explained if the intermediate state of regulated actin has a unique function. The intermediate state appears to have an ability to accelerate the rate of ATP hydrolysis by myosin that exceeds that of the inactive state.

An analysis of the primary structure of the actin-binding protein fesselin revealed it to be the avian homologue of mammalian synaptopodin 2 [Schroeter, Beall, Heid, and Chalovich (2008) Biochem. Biophys. Res. Commun. 371, 582–586]. We isolated two synaptopodin 2 isoforms from rabbit stomach that corresponded to known types of human synaptopodin 2. The purification scheme used was that developed for avian fesselin. These synaptopodin 2 forms shared several key functions with fesselin. Both avian fesselin and mammalian synaptopodin 2 bound to Ca2+–calmodulin, α-actinin and smooth-muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca2+–calmodulin-dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of α-actinin, to polymerize actin in a Ca2+–calmodulin-dependent manner, or to bind to Ca2+–calmodulin or myosin. These properties are consistent with the proposed function of synaptopodin 2 in organizing the cytoskeleton.

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and alpha-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.

Alterations in the troponin complex can lead to increases or decreases in contractile activity. Most mutations of troponin that cause hypertrophic cardiomyopathy increase the activity of cardiac muscle fibers. In at least some cases these mutants stabilize the active state of regulated actin. In contrast, phosphorylation of troponin I at residues 43, 45, and 144 inhibits muscle contractility. To determine if alterations of troponin I that reduce activity do stabilize the inactive state of actin, we introduced negative charges at residues 43, 45, and 144 of troponin I to mimic a constitutively phosphorylated state. At saturating calcium, all mutants decreased ATPase rates relative to wild-type actin-tropomyosin-troponin. Reduced activation of ATPase activity was seen with a single mutation at S45E and was not further altered by mutating the other two sites. In the presence of low concentrations of NEM-S1, wild-type troponin was more active than the mutants. At high NEM-S1, the rates of wild-type and mutants approached the same limiting value. Changes in Ca2+ affinity also support the idea that the equilibrium between states of actin-tropomyosin-troponin was shifted to the inactive state by mutations that mimic troponin I phosphorylation.

Fesselin is a heat stable proline-rich actin binding protein. The stability, amino acid composition, and ability to bind to several proteins suggested that fesselin may be unfolded under native conditions. While the complete sequence of fesselin is unknown an analysis of a closely related protein, synaptopodin 2 from Gallus gallus, indicates that fesselin consists of a series of unstructured regions interspersed between short folded regions. To determine if fesselin is natively unfolded, we compared fesselin to a known globular protein (myosin S1) and a known unfolded protein Cad22 (the COOH terminal 22 kDa fragment of caldesmon). Fesselin, and Cad22, had larger Stokes radii than globular proteins of equivalent mass. The environments of tryptophan residues of fesselin and Cad22 were the same in the presence and absence of 6 M guanidine hydrochloride. Fesselin had a circular dichroism spectrum that was primarily random coil. Changes in pH over the range of 1.5-11.5 did not alter that spectrum. Increasing the temperature to 85 degrees C caused an increase in the degree of secondary structure. Calmodulin binding to fesselin altered the environment of the tryptophan residues so that they became less sensitive to the quencher acrylamide. These results show that fesselin is a natively unfolded protein.

Actin–tropomyosin–troponin has three structural states, but the functional properties of regulation can be explained with models having two functional states. As a step towards assigning functional properties to all the structural states, we examined fluorescent probes that monitor changes in troponin and tropomyosin. Tropomyosin labeled with pyrene–iodoacetamide is thought to reflect the transition to the most active state, whereas N-((2-iodoacetoxy)ethyl)-N-methyl)-amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I is thought to monitor the transition to any state other than the inactive state. The fraction of actin in an active state determined from pyrene excimer fluoresecence agreed with that calculated from light-scattering measurements of myosin subfragment 1 (S1)–ADP to regulated actin in both the presence and absence of Ca2+ over a range of ionic strength conditions. The only exceptions were conditions where the binding of S1–ADP to actin was too strong to measure accurately. Pyrene–tropomyosin excimer fluorescence was Ca2+ dependent and so reflected the change in population caused by both Ca2+ binding and S1–ADP binding. Pyrene labeling of tropomyosin did not cause a large perturbation of the transition among states of regulated actin. Using pyrene–tropomyosin fluorescence we were able to extend the ionic strength dependence of the parameters describing the co-operativity of binding of S1–ADP to actin as low as 0.1 m. The probes on tropomyosin and troponin I had different responses to Ca2+ and S1–ADP binding. These different sensitivities can be explained by an intermediate between the inactive and active states of regulated actin.

Fesselin (or avian synaptopodin 2) is an actin binding protein that nucleates actin filament formation (Leinweber et al. 1999; Beall et al. 2001) and bundles actin filaments (Schroeter et al. 2013). The nucleating activity is inhibited by Ca2+-calmodulin (Schroeter et al. 2004). Fesselin also binds myosin with an affinity of 2 x 106 M−1 at 50 mM ionic strength (Schroeter & Chalovich 2005). That binding increases the length and width of myosin filaments, and reduces the rate of dissociation of myosin filaments and actin-myosin complexes by ATP (Kingsbury et al. 2013). Fesselin labeled with IANBD produced a fluorescence increase upon binding to smooth muscle myosin or S1. The apparent rate constant for the fluorescence change increased with increasing concentrations of myosin or S1 in a hyperbolic fashion (105 mM ionic strength, 10 deg C). Binding to myosin began with the rapid formation of a low affinity intermediate followed by a second step having an observed rate constant (k2 + k-2) of 30/sec. Several observations suggested that the binding of fesselin to myosin was affected by calmodulin and calcium. The apparent rate constants for both processes in the binding of IANBD-fesselin to S1 were 2-fold faster in the absence of calcium. Binding of IANBD fesselin to intact myosin was complex in the presence of calcium with a rapid increase in fluorescence followed by a slower decrease; that behavior was not observed in the presence of calcium but absence of calmodulin. Ca2+-calmodulin may play roles in the formation of actin filaments, myosin filaments and actin-myosin complexes through fesselin.

Fesselin is an actin binding protein that bundles actin filaments and accelerates nucleation of actin polymerization. The effect of fesselin on actin polymerization is regulated by Ca(++)-calmodulin. Because actin filaments serve both structural and contractile functions we also examined the effect of fesselin on activation of myosin S1 ATPase activity. Fesselin inhibited the activation of S1-catalyzed ATP hydrolysis in a similar manner in both the presence and absence of tropomyosin. This inhibition was unaffected by Ca(++)-calmodulin. Fesselin inhibited the binding of myosin-S1 to actin during steady-state ATP hydrolysis. Fesselin also displaced caldesmon from actin. S1 displaced fesselin from actin in the absence of nucleotide when the affinity of S1 for actin was much greater than the affinity of fesselin for actin. It is likely that fesselin and S1 share common binding sites on F-actin. We also observed that fesselin could bind to smooth muscle myosin with muM affinity. Fesselin shares some similarities to caldesmon in binding to several other proteins and having multiple potential functions.

The complex of tropomyosin and troponin binds to actin and inhibits activation of myosin ATPase activity and force production of striated muscles at low free Ca2+ concentrations. Ca2+ stimulates ATP activity and at subsaturating actin concentrations the binding of NEM-modified S1 to actin– tropomyosin–troponin increases the rate of ATP hydrolysis even further. We show here that the ∆14 mutation of troponin T associated with familial hypertrophic cardiomyopathy results in an increase in ATPase rate like that seen with wild-type troponin in the presence of NEM-S1. The enhanced ATPase activity was not due to a decreased incorporation of mutant troponin T with roponin I and troponin C to form an active troponin complex. The activating effect was more prominent with a hybrid troponin (skeletal TnI TnC and cardiac TnT) than with all cardiac troponin. Thus it appears that changes in the troponin–troponin contacts that result from mutations or from forming hybrids stabilize a more active state of regulated actin. An analysis of the effect of the ∆14 mutation on the equilibrium binding of S1-ADP to actin was consistent with stabilization of an active state of actin. This change in activation may be important in the development of cardiac disease. Originally published Biochemistry Vol. 43 No. 48 Dec 2004

There is no consensus on the mechanism of inhibition of actin-myosin ATPase activity by caldesmon. Various models are based on different assumptions for the number of actin monomers that constitute a caldesmon binding site. Differences in binding behavior may be due to variations in the assay, the range of caldesmon concentrations, the type of caldesmon, and the method of data analysis used. We have evaluated these factors by measuring binding in the presence and absence of tropomyosin with both intact caldesmon and a recombinant 35 kDa actin binding fragment and with actin initially in the polymerized state or monomeric state. In all cases caldesmon binding could be simulated with a model having one class of binding sites. However, the number of actin monomers constituting a site was variable. Binding to F-actin at 165 mM ionic strength was best described with 7 actin monomers per site. When caldesmon bound to actin during the polymerization of G-actin, the size of the binding site was 3. Binding of the expressed truncated fragment, Cad35, could be described with 3 monomers per site. A simple interpretation of the data is that caldesmon binds tightly to 2-3 actin monomers. Additional parts of caldesmon bind less tightly to actin, causing caldesmon to cover approximately 7 actin monomers. The appendix contains an analysis of several binding curves with multiple binding site models. There is no compelling evidence for two classes of binding sites.

Several laboratories have reported cooperative binding of S1 to actin in the presence of caldesmon. This cooperative binding has been interpreted with a model similar to that proposed for the binding of S 1 to regulated actins in which the binding affinity of S 1 is controlled by the position of the tropomyosin filaments. In a recent paper [Sen, A., Chen, Y., Yan, B., and Chalovich, J. M. (2001) Biochemistry 40, 5757-64], we showed qualitatively that Sl binding resulted in rapid dissociation of caldesmon from actin or actin-tropomyosin. This suggests that the cooperativity observed in the case of caldesmon is not due to a conformational change in actin-caldesmon but to the displacement of caldesmon. We show in this paper that the pure competitive binding model, in which both Sl and caldesmon are competing for the same binding sites on actin, can simulate quantitatively the effect of caldesmon on both the equilibrium and the kinetics of Sl binding to actin. This model successfully predicts an apparent cooperativity for the binding of S 1 to actin-caldesmon without the need to assume multiple actin-caldesmon structures and produces a decreased rate of S 1 binding to actin in the presence of caldesmon. This suggests that the inhibitory action of caldesmon on the actin-activated ATPase activity of myosin in solution and on the generation of active force in a contracting muscle may be simply due to the blocking of myosin binding sites on actin by caldesmon.

Actin stimulates the ATPase activity of myosin-S1. The regulatory proteins tropomyosin and troponin prevent this stimulation at conditions of low Ca2+ and low high-affinity myosin binding. At saturating Ca2+ and with strong actin-binding species of myosin (S1-ADP or NEM-S1) the enhancement of ATPase activity exceeds that seen in the absence of regulatory proteins. Inhibition and potentiation of actin activated ATPase activity are dependent on the position of tropomyosin on actin filaments. One position of tropomyosin (M state) leads to high activity whereas the other two positions (B and the intermediate C state) are inactive.We recently showed that the C-terminal 14 residues of troponin T are critical for formation of the inactive B state at low Ca2+. Furthermore, that same region of troponin T limits the formation of the M state at saturating Ca2+. Our present goal is to map the C-terminal region of troponin T to determine if specific residues in the sequence are responsible for each of these activities. The B state was monitored by changes in acrylodan tropomyosin fluorescence and by the rate of rigor S1 binding to regulated actin. The M state was monitored by an ATPase assay. Stepwise deletions within the C-terminal 14 amino acids of Troponin T progressively reduced the population of the inactive B state at low Ca2+. In contrast, the population of the M state was progressively increased at high Ca2+. The net positive charge of the last 14 residues appeared to be a key determinant of activity. Conclusion: The C-terminal region of troponin T plays essential roles in inactivation and in limiting activation. Both of these activities require basic amino acid residues within the COOH terminus.

It was previously shown that a one-dimensional Ising model could successfully simulate the equilibrium binding of myosin S1 to regulated actin filaments (T. L. Hill, E. Eisenberg and L. Greene, Proc. Natl. Acad. Sci. U.S.A. 77:3186–3190, 1980). However, the time course of myosin S1 binding to regulated actin was thought to be incompatible with this model, and a three-state model was subsequently developed (D. F. McKillop and M. A. Geeves, Biophys. J. 65:693–701, 1993). A quantitative analysis of the predicted time course of myosin S1 binding to regulated actin, however, was never done for either model. Here we present the procedure for the theoretical evaluation of the time course of myosin S1 binding for both models and then show that 1) the Hill model can predict the "lag" in the binding of myosin S1 to regulated actin that is observed in the absence of Ca ++ when S1 is in excess of actin, and 2) both models generate very similar families of binding curves when [S1]/[actin] is varied. This result shows that, just based on the equilibrium and pre-steady-state kinetic binding data alone, it is not possible to differentiate between the two models. Thus, the model of Hill et al. cannot be ruled out on the basis of existing pre-steady-state and equilibrium binding data. Physical mechanisms underlying the generation of the lag in the Hill model are discussed.

The troponin complex in a muscle fiber can be replaced with exogenous troponin by using a gentle exchange procedure in which the actin–tropomyosin complex is never devoid of a full complement of troponin (Brenner et al. (1999) Biophys J 77: 2677–2691). The mechanism of this exchange process and the factors that influence this exchange are poorly understood. In this study, the exchange process has now been examined in myofibrils and in solution. In myofibrils under rigor conditions, troponin exchange occurred preferentially in the region of overlap between actin and myosin when the free Ca2+ concentration was low. At higher concentrations of Ca2+, the exchange occurred uniformly along the actin. Ca2+ also accelerated troponin exchange in solution but the effect of S1 could not be confirmed in solution experiments. The rate of exchange in solution was insensitive to moderate changes in pH or ionic strength. Increasing the temperature resulted in a two-fold increase in rate with each 10°C increase in temperature. A sequential two step model of troponin binding to actin–tropomyosin could simulate the observed association and dissociation transients. In the absence of Ca2+ or rigor S1, the following rate constants could describe the binding process: k 1 = 7.12 μM−1s−1, k −1 = 0.65 s−1, k 2 = 0.07 s−1, k −2 = 0.0014 s−1. The slow rate of detachment of troponin from actin (k −2) limits the rate of exchange in solution and most likely contributes to the slow rate of exchange in fibers.

The purpose of this study was to address the paradox of calponin localization with alpha-actinin and filamin, two proteins with tandem calponin homology (CH) domains, by determining the effect of these proteins on the binding of calponin to actin. The results show that actin can accommodate near-saturating concentrations of either calponin and alpha-actinin or calponin and filamin with little change or no change in ligand affinity. Little direct interaction occurred between alpha-actinin and calponin in the absence of actin, so this effect is not likely to explain the co-distribution of these proteins. Calponin, like alpha-actinin, induced elastic gel formation when added to actin. When alpha-actinin was added to newly formed calponin/actin gels, no change was seen in the mechanical properties of the gel compared to calponin and actin alone. However, when calponin was added to newly formed alpha-actinin/actin gels, the resulting gel was much stronger than the gels formed by either ligand alone. Furthermore, gels formed by the addition of calponin to alpha-actinin/actin exhibited a phenomenon known as strain hardening, a characteristic of mechanically resilient gels. These results add weight to the concept that one of the functions of calponin is to stabilize the actin cytoskeleton.

A novel actin binding protein has been isolated from chicken gizzard muscle. When isolated, a pair of proteins with apparent molecular weights of 79 kDa and 103 kDa are obtained; both proteins have a pI near 9.3. Peptide mapping indicates that these proteins are related. Antibodies against this protein cross-reacted with proteins from other smooth muscle containing tissues as well as skeletal and heart muscle. Traces of cross-reactive material were also detected in brain and kidney tissue. The affinity of this protein for actin is ca. 1 × 106 M−1. Interestingly, this actin binding protein is a potent actin-bundling agent. A partial sequence analysis confirmed that there were no previously reported homologues in smooth muscle. However, considerable homology was found with the protein synaptopodin that is found in nervous tissue and kidney but is absent from muscle tissue. It is likely that the new protein is a member of the synaptopodin family. We call the smooth muscle actin binding protein fesselin.

Intact caldesmon and particularly the actin-binding C-terminal fragment (20-kDa) of caldesmon have been shown in skeletal muscle fibers to selectively displace low affinity, weakly bound cross-bridges from actin without significantly altering the actin attachment of force producing, strong binding cross-bridges (Brenner et al., 1991; Kraft et al., 1995a). However, the sarcomeric distribution and the specific binding of externally added caldesmon to the myofilaments of skeletal muscle fibers was not known. It was e.g., unclear whether caldesmon binds along actin in a manner similar to tropomyosin or whether it also binds to myosin. In this study, we determined the binding pattern of exogenously added intact caldesmon and its C-terminal 20-kDa fragment, respectively, in MgATP-relaxed rabbit skeletal muscle fibers using electron (EM) and confocal fluorescence microscopy (CFM). EM showed that similar to what has been demonstrated earlier for smooth muscle thin filaments (Lehman et al., 1989), intact caldesmon binds periodically every 38nm along the thin filaments. CFM revealed that rhodamine-labeled intact caldesmon and the 20-kDa caldesmon fragment bind along nearly the entire length of the thin filaments. A portion of the I-band near the Z-line appears unlabeled, both when equilibrated at normal and long sarcomere lengths. The width of the unlabeled region seems to depend on ionic strength. The 20-kDa C-terminal caldesmon fragment binds in essentially the same pattern as intact caldesmon. This indicates that the high fluorescence intensity in the overlap region seen with intact caldesmon does not depend on caldesmon binding to myosin. X-ray diffraction was used to monitor the effects on filament lattice. Intact caldesmon at >0.3mg/ml induced disorder in the myofilament lattice. No such disordering was observed, however, when fibers were equilibrated with up to 0.8mg/ml of the 20-kDa caldesmon fragment.

Fesselin or avian synaptopodin 2 stimulates actin polymerization in a Ca2+-calmodulin dependent manner. Fesselin binding to F-actin inhibits myosin S1 binding and yet fesselin binds with moderate affinity to smooth muscle myosin. These properties suggest that fesselin could tether actin and myosin together in an inactive complex as caldesmon does. This possibility was tested by observing the effect of fesselin on the rate of dissociation of actin-myosin by ATP in a stopped-flow device. Dissociation was measured by light scattering (a measure of particle size) and by pyrene actin fluorescence (a specific measure of actin-myosin binding). Fesselin reduced the multi-exponential rates of change of light scattering and pyrene fluorescence in concentration dependent manners. Each light scattering trace had a rapid initial transition that was not present in pyrene fluorescence traces. That rapid light scattering change was likely due to dissociation of filamentous myosin. The reduction in rate of that rapid process could mean that fesselin alters the structure and dissociation kinetics of smooth myosin filaments. We examined changes in smooth muscle myosin with electron microscopy. ATP caused dissociation of myosin filaments in both the presence and absence of fesselin. In the absence of ATP, fesselin increased the size of myosin filaments. Furthermore, the filaments were arranged in parallel arrays. These results indicate that fesselin cross-links myosin to actin and also organizes myosin filaments in solution.

Radial equilibrium lengths of the weakly attached, force-generating, and rigor cross-bridges are determined by recording their resistance to osmotic compression. Radial equilibrium length is the surface-to-surface distance between myosin and actin filaments at which attached cross-bridges are, on average, radially undistorted. We previously proposed that differences in the radial equilibrium length represent differences in the structure of the actomyosin cross-bridge. Until now the radial equilibrium length had only been determined for various strongly attached cross-bridge states and was found to be distinct for each state examined. In the present work, we demonstrate that weakly attached cross-bridges, in spite of their low affinity for actin, also exert elastic forces opposing osmotic compression, and they are characterized by a distinct radial equilibrium length (12.0 nm vs. 10.5 nm for force-generating and 13.0 nm for rigor cross-bridge). This suggests significant differences in the molecular structure of the attached cross-bridges under these conditions, e.g., differences in the shape of the myosin head or in the docking of the myosin to actin. Thus, the present finding supports our earlier conclusion that there is a structural change in the attached cross-bridge associated with the transition from a weakly bound configuration to the force-generating configuration. The implications for imposing spatial constraints on modeling actomyosin interaction in the filament lattice are discussed.

The slowly hydrolyzed ATP analog adenosine 5'-(gamma-thiotriphosphate) (ATP gamma S) has been used in many studies of the muscle motor protein myosin in order to form a stable "weak binding" state analogous to the actin-S1-ATP complex However, the results from studies using ATP gamma S do not always agree with the results of experiments using ATP. The binding of myosin subfragment-1-ATP gamma S to actin has now been studied in some detail to determine its relationship to the actin-S1-ATP state. The binding of myosin subfragment-1-ATP gamma S to actin-troponin-tropomyosin is similar in affinity to the binding of myosin subfragment-1-ATP. Like myosin subfragment-1-ATP, the binding is not Ca(2+)-dependent, and most importantly, myosin subfragment-1-ATP gamma S does not stabilize the active configuration of actin-troponin-tropomyosin. Thus, myosin subfragment-1-ATP gamma S is an analog of myosin subfragment-1-ATP but must be used with caution for two reasons: (1) The binding of ATP gamma S to regulated actomyosin subfragment-1 is Ca(2+)-sensitive, and errors can be made in the interpretation of results if proteins are not fully saturated with nucleotide and a mixture of weak and strong binding states is present. (2) At the high concentrations of myosin subfragment-1 used in some experiments, significant amounts of ADP may form.(ABSTRACT TRUNCATED AT 250 WORDS)

Calponin, a protein isolated from smooth muscle and nonmuscle cells, has previously been shown to inhibit the actin-activated ATPase activity of myosin. Reports of the stoichiometry of binding range from 1 calponin per actin to 1 calponin per 3 actin monomers. We now report a detailed study of the binding of [14C]iodoacetamide-labeled calponin to actin. The labeling procedure did not significantly alter the binding constant of calponin to actin. The stoichiometry of binding was variable and dependent on ionic strength. Below 110 mM ionic strength, the stoichiometry of binding was 1:1. As the ionic strength was increased above 110 mM ionic strength, the stoichiometry shifted from 1:1 to 1 calponin per 2 actin monomers. At physiological ionic strength, the binding exhibited a small degree of positive cooperativity and was adequately described by a single class of binding sites with an association constant of 6 x 10(6) M-1. The affinity decreased to 20% of this value in the presence of ATP. Irrespective of the ionic strength, actin formed bundles when saturation with calponin exceeded about 30%. Measurements of the rate of association were complicated by this bundling, but the upper limit for this reaction was placed at 10(6) M-1 s-1. The addition of calponin to actin-caldesmon complexes caused displacement of the caldesmon.

Previously we showed that stiffness of relaxed fibers and active force generated in single skinned fibers of rabbit psoas muscle are inhibited in parallel by actin-binding fragments of caldesmon, an actin-associated protein of smooth muscle, under conditions in which a large fraction of cross-bridges is weakly attached to actin (ionic strength of 50 mM and temperature of 5 degrees C). These results suggested that weak cross-bridge attachment to actin is essential for force generation. The present study provides evidence that this is also true for physiological ionic strength (170 mM) at temperatures up to 30 degrees C, suggesting that weak cross-bridge binding to actin is generally required for force generation. In addition, we show that the inhibition of active force is not a result of changes in cross-bridge cycling kinetics but apparently results from selective inhibition of weak cross-bridge binding to actin. Together with our previous biochemical, mechanical, and structural studies, these findings support the proposal that weak cross-bridge attachment to actin is an essential intermediate on the path to force generation and are consistent with the concept that isometric force mainly results from an increase in strain of the attached cross-bridge as a result of a structural change associated with the transition from a weakly bound to a strongly bound actomyosin complex. This mechanism is different from the processes responsible for quick tension recovery that were proposed by Huxley and Simmons (Proposed mechanism of force generation in striated muscle. Nature. 233:533-538.) to represent the elementary mechanism of force generation.