Publications by authors named "Joseph M Chalovich"

46 Publications

Caldesmon ablation in mice causes umbilical herniation and alters contractility of fetal urinary bladder smooth muscle.

J Gen Physiol 2021 Jul 11;153(7). Epub 2021 Jun 11.

Institute of Vegetative Physiology, Center of Physiology, Faculty of Medicine, University of Cologne, Cologne, Germany.

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Cald1-/- compared with Cald1+/+ skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KCl-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cald1-/- than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure.
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http://dx.doi.org/10.1085/jgp.202012776DOI Listing
July 2021

Structure of the shutdown state of myosin-2.

Nature 2020 12 2;588(7838):515-520. Epub 2020 Dec 2.

The Astbury Centre for Structural and Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.

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 heads, which prevents filament formation and inactivates the motors. 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 Å 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.
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http://dx.doi.org/10.1038/s41586-020-2990-5DOI Listing
December 2020

The Positively Charged C-Terminal Region of Human Skeletal Troponin T Retards Activation and Decreases Calcium Sensitivity.

Biochemistry 2020 11 19;59(43):4189-4201. Epub 2020 Oct 19.

Department of Biochemistry & Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina 27834, United States.

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 Ca 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 <10 nM free Ca. 251C-HAHA-TnT and Δ16-TnT mutants greatly increased ATPase activation at 0.2 mM Ca, even without high-affinity cross-bridge binding. They also shifted the force-pCa curve of muscle fibers to lower Ca by 0.8-1.2 pCa units (the larger shift for 251C-HAHA-TnT). Shifts in force-pCa were maintained in the presence of -aminoblebbistatin. The effects of modification of the C-terminal region of TnT on the kinetics of S1 binding to actin were somewhat different from those observed earlier with the cardiac analogue. In general, the C-terminal region of human skeletal TnT is critical to regulation, just as it is in the cardiac system, and is a potential target for modulating activity.
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http://dx.doi.org/10.1021/acs.biochem.0c00499DOI Listing
November 2020

Eliminating the First Inactive State and Stabilizing the Active State of the Cardiac Regulatory System Alters Behavior in Solution and in Ordered Systems.

Biochemistry 2020 09 9;59(37):3487-3497. Epub 2020 Sep 9.

Department of Biochemistry & Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina, United States.

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 and to eliminate one of the inactive states at low Ca. 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, full activation with Δ14-TnT·A8V-TnC decreased the apparent for actin-activated ATPase activity compared to bare actin filaments. Rates of in vitro motility increased at both high and low Ca with Δ14-TnT; the maximum shortening speed at high Ca increased 1.8-fold. Cardiac muscle preparations exhibited increased Ca 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 and Δ14-TnT·A8V-TnC confirmed and extended several earlier observations using other means of reaching full activation. Furthermore, at low Ca, 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.
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http://dx.doi.org/10.1021/acs.biochem.0c00430DOI Listing
September 2020

Cycling Cross-Bridges Contribute to Thin Filament Activation in Human Slow-Twitch Fibers.

Front Physiol 2020 24;11:144. Epub 2020 Mar 24.

Institute of Vegetative Physiology, University of Cologne, Cologne, Germany.

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 ( = 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.
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http://dx.doi.org/10.3389/fphys.2020.00144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105683PMC
March 2020

Basic residues within the cardiac troponin T C terminus are required for full inhibition of muscle contraction and limit activation by calcium.

J Biol Chem 2019 12 11;294(51):19535-19545. Epub 2019 Nov 11.

Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina 27834

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 and for limiting its activation at saturating Ca Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca-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 Ca 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 Ca 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 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.
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http://dx.doi.org/10.1074/jbc.RA119.010966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926443PMC
December 2019

Stepwise C-Terminal Truncation of Cardiac Troponin T Alters Function at Low and Saturating Ca.

Biophys J 2018 08 12;115(4):702-712. Epub 2018 Jul 12.

Department of Biochemistry, Brody School of Medicine, East Carolina University, Greenville, North Carolina. Electronic address:

Activation of striated muscle contraction occurs in response to Ca 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. 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 effects. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca. Changes in acrylodan tropomyosin fluorescence and the degree of Ca stimulation of the rate of binding of rigor myosin subfragment 1 to pyrene-labeled actin-tropomyosin-troponin were measured at low Ca. 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. 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 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.
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http://dx.doi.org/10.1016/j.bpj.2018.06.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6104287PMC
August 2018

Obituary Bernhard Brenner.

J Muscle Res Cell Motil 2017 Aug;38(3-4):269-270

Medizinische Hochschule Hannover, Hannover, Germany.

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http://dx.doi.org/10.1007/s10974-017-9488-2DOI Listing
August 2017

Troponin C Mutations Partially Stabilize the Active State of Regulated Actin and Fully Stabilize the Active State When Paired with Δ14 TnT.

Biochemistry 2017 06 31;56(23):2928-2937. Epub 2017 May 31.

Department of Biochemistry & Molecular Biology, Brody School of Medicine at East Carolina University , Greenville, North Carolina 27858, United States.

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 and activating cross-bridges (i.e., S1-ADP or rigor S1). Binding of activating species of myosin to actin at a saturating Ca concentration stabilizes the most active state (M state) of the actin-tropomyosin-troponin complex (regulated actin). Ca binding alone produces partial stabilization of the active state. The extent of stabilization at a saturating Ca 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 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 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 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. Because Ca 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.
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http://dx.doi.org/10.1021/acs.biochem.6b01092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448410PMC
June 2017

Commentary: Effect of Skeletal Muscle Native Tropomyosin on the Interaction of Amoeba Actin with Heavy Meromyosin.

Front Physiol 2016 31;7:377. Epub 2016 Aug 31.

Department of Chemistry, East Carolina University Greenville, NC, USA.

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http://dx.doi.org/10.3389/fphys.2016.00377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006597PMC
September 2016

The Cardiomyopathy Mutation, R146G Troponin I, Stabilizes the Intermediate "C" State of Regulated Actin under High- and Low-Free Ca(2+) Conditions.

Biochemistry 2016 08 3;55(32):4533-40. Epub 2016 Aug 3.

Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University , Greenville, North Carolina 27834, United States.

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.
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http://dx.doi.org/10.1021/acs.biochem.5b01359DOI Listing
August 2016

Avian synaptopodin 2 (fesselin) stabilizes myosin filaments and actomyosin in the presence of ATP.

Biochemistry 2013 Oct 18;52(43):7641-7. Epub 2013 Oct 18.

Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University , 600 Moye Boulevard, Greenville, North Carolina 27834-4300, United States.

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.
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http://dx.doi.org/10.1021/bi401013gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859813PMC
October 2013

Organization of F-actin by Fesselin (avian smooth muscle synaptopodin 2).

Biochemistry 2013 Jul 9;52(29):4955-61. Epub 2013 Jul 9.

Institute of Vegetative Physiology, University of Cologne , Robert Koch Strasse 39, D-50931 Cologne, Germany.

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.
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http://dx.doi.org/10.1021/bi4005254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842371PMC
July 2013

Structural dynamics of troponin I during Ca2+-activation of cardiac thin filaments: a multi-site Förster resonance energy transfer study.

PLoS One 2012 5;7(12):e50420. Epub 2012 Dec 5.

Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America.

A multi-site, steady-state Förster resonance energy transfer (FRET) approach was used to quantify Ca(2+)-induced changes in proximity between donor loci on human cardiac troponin I (cTnI), and acceptor loci on human cardiac tropomyosin (cTm) and F-actin within functional thin filaments. A fluorescent donor probe was introduced to unique and key cysteine residues on the C- and N-termini of cTnI. A FRET acceptor probe was introduced to one of three sites located on the inner or outer domain of F-actin, namely Cys-374 and the phalloidin-binding site on F-actin, and Cys-190 of cTm. Unlike earlier FRET analyses of protein dynamics within the thin filament, this study considered the effects of non-random distribution of dipoles for the donor and acceptor probes. The major conclusion drawn from this study is that Ca(2+) and myosin S1-binding to the thin filament results in movement of the C-terminal domain of cTnI from the outer domain of F-actin towards the inner domain, which is associated with the myosin-binding. A hinge-linkage model is used to best-describe the finding of a Ca(2+)-induced movement of the C-terminus of cTnI with a stationary N-terminus. This dynamic model of the activation of the thin filament is discussed in the context of other structural and biochemical studies on normal and mutant cTnI found in hypertrophic cardiomyopathies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050420PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515578PMC
June 2013

Thermodynamics and molecular dynamics simulations of calcium binding to the regulatory site of human cardiac troponin C: evidence for communication with the structural calcium binding sites.

J Biol Inorg Chem 2013 Jan 31;18(1):49-58. Epub 2012 Oct 31.

Department of Chemistry, East Carolina University, 300 Science and Technology, Greenville, NC 27858, USA.

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.
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http://dx.doi.org/10.1007/s00775-012-0948-2DOI Listing
January 2013

The C-terminus of troponin T is essential for maintaining the inactive state of regulated actin.

Biophys J 2012 Jun 5;102(11):2536-44. Epub 2012 Jun 5.

Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, USA.

Striated muscle contraction is regulated by the actin binding proteins tropomyosin and troponin. Defects in these proteins lead to myopathies and cardiomyopathies. Deletion of the 14 C-terminal residues of cardiac troponin T leads to hypertrophic cardiomyopathy. We showed earlier that regulated actin containing Δ14 TnT was more readily activated than wild-type regulated actin. We suggested that the equilibria among the inactive (blocked), intermediate (closed or calcium), and active (open or myosin) states was shifted to the active state. We now show that, in addition, such regulated actin filaments cannot enter the inactive or blocked state. Regulated actin containing Δ14 TnT had ATPase activities in the absence of Ca2+ that were higher than wild-type filaments but far below the fully active rate. The rapid dissociation of S1-ATP from regulated actin filaments containing Δ14 TnT and acrylodan-labeled tropomyosin did not show the fluorescence increase characteristic of moving to the inactive state. Replacing wild-type TnI with S45E TnI, that favors the inactive state, did not restore the fluorescence change. We conclude that TnT has a previously unrecognized role in forming the inactive state of regulated actin.
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http://dx.doi.org/10.1016/j.bpj.2012.04.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368147PMC
June 2012

Disease causing mutations of troponin alter regulated actin state distributions.

J Muscle Res Cell Motil 2012 Dec 8;33(6):493-9. Epub 2012 Jun 8.

Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC, USA.

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.
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http://dx.doi.org/10.1007/s10974-012-9305-xDOI Listing
December 2012

Michael Bárány: a recollection.

J Muscle Res Cell Motil 2012 Dec 27;33(6):373-6. Epub 2012 Apr 27.

Department of Biochemistry & Molecular Biology, East Carolina University School of Medicine, 5E-122 Brody Building, 600 Moye Blvd., Greenville, NC 27834, USA.

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.
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http://dx.doi.org/10.1007/s10974-012-9295-8DOI Listing
December 2012

Acrylodan-labeled smooth muscle tropomyosin reports differences in the effects of troponin and caldesmon in the transition from the active state to the inactive state.

Biochemistry 2011 Jul 14;50(27):6093-101. Epub 2011 Jun 14.

Brody School of Medicine at East Carolina University, 5E-122 Brody Medical Sciences Building, Greenville, North Carolina 27834, USA.

Changes in the orientation of tropomyosin on actin are important for the regulation of striated muscle contraction and could also be important for smooth muscle regulation. We showed earlier that acrylodan-labeled skeletal muscle tropomyosin reports the kinetics of the reversible transitions among the active, intermediate, and inactive states when S1 is rapidly detached from actin-tropomyosin. We now show that acrylodan-labeled smooth muscle tropomyosin reports similar transitions among states of actin-tropomyosin. When S1 was rapidly detached from actin-smooth muscle tropomyosin, there was a rapid decrease in acrylodan-tropomyosin fluorescence as the intermediate state became populated. The rate constant for this process was >600 s(-1) at temperatures near 5 °C. In the presence of skeletal troponin and EGTA, the decrease in fluorescence was followed by the redevelopment of fluorescence as the inactive state became populated. The apparent rate constant for the fluorescence increase was 14 s(-1) at 5 °C. Substituting smooth muscle caldesmon for skeletal muscle troponin produced a similar decrease and re-increase in fluorescence, but the apparent rate constant for the increase was >10 times that observed with troponin. Furthermore, the fluorescence increase was correlated with an increase in the extent of caldesmon attachment as S1-ATP dissociated. Although the measured rate constant appeared to reflect the rate-limiting transition for inactivation, it is unclear if the fluorescence change resulted from caldesmon binding, the movement of tropomyosin over actin, or both.
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http://dx.doi.org/10.1021/bi200288cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145316PMC
July 2011

Role of the tail in the regulated state of myosin 2.

J Mol Biol 2011 May 23;408(5):863-78. Epub 2011 Mar 23.

Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation.
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http://dx.doi.org/10.1016/j.jmb.2011.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776433PMC
May 2011

A computational and experimental approach to investigate bepridil binding with cardiac troponin.

J Phys Chem B 2011 Mar 18;115(10):2392-400. Epub 2011 Feb 18.

Department of Chemistry, Brody School of Medicine, East Carolina University, Greenville, North Carolina 27858, USA.

Cardiac troponin is a Ca(2+)-dependent switch for the contraction in heart muscle and a potential target for drugs in the therapy of heart failure. Bepridil is a drug that binds to troponin and increases calcium sensitivity of muscle contraction. Because bepridil has been well studied, it is a good model for analysis by computational and experimental methods. Molecular dynamics (MD) simulations were performed on troponin complexes of different sizes in the presence and absence of bepridil bound within the hydrophobic pocket at the N-terminal domain of troponin C. About 100 ns of simulation trajectory data were generated, which were analyzed using cross-correlation analyses and MMPBSA and MMGBSA techniques. The results indicated that bepridil binding within the hydrophobic pocket of cardiac TnC decreases the interaction of TnC with TnI at both the N-domain of TnC and the C-domain of TnC, and decreases the correlations of motions among the segments of the troponin subunits. The estimated calcium-binding affinities using MMPBSA showed that bepridil has a sensitizing effect for the isolated system of TnC, but loses this effect for the complex. Our experimental measurements of calcium dissociation rates were consistent with that prediction. We also observed that while bepridil enhanced the troponin-tropomyosin-actin-activated ATPase activity of myosin S1 at low calcium concentrations it was slightly inhibitory at high calcium concentrations. Bepridil increases the ATPase activity and force generation in muscle fibers, but its effects appear to depend on the concentration of calcium.
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http://dx.doi.org/10.1021/jp1094504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807686PMC
March 2011

Several cardiomyopathy causing mutations on tropomyosin either destabilize the active state of actomyosin or alter the binding properties of tropomyosin.

Biochem Biophys Res Commun 2011 Mar 3;406(1):74-8. Epub 2011 Feb 3.

Department of Biochemistry and Molecular Biology, 600 Moye Blvd., Brody School of Medicine at East Carolina University, Greenville, NC 27834, USA.

We examined the cardiomyopathy-causing tropomyosin mutations E180G, D175N, and V95A to determine their effects on actomyosin regulation. V95A reduced the ATPase rate when filaments were saturated with regulatory proteins both in the presence and absence of calcium, indicating either a stabilization of the inactive state or an inability to fully populate the active state. Effects of E180G and D175N were more complex. These two mutations increased ATPase rates at sub-saturating concentrations of troponin and tropomyosin as compared to wild type tropomyosin. At higher concentrations of regulatory proteins, ATPase rates became similar to wild type. Normal activation was achieved with the tight-binding myosin analog N-ethylmaleimide-S1, at saturating regulatory protein concentrations. These results suggest that the E180G and D175N mutations reduce the affinity of tropomyosin for actin and also destabilize troponin binding to the actin thin filaments.
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http://dx.doi.org/10.1016/j.bbrc.2011.01.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057449PMC
March 2011

Synaptopodin family of natively unfolded, actin binding proteins: physical properties and potential biological functions.

Biophys Rev 2010 Dec 20;2(4):181-189. Epub 2010 Nov 20.

Department of Physiology, University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, Germany.

The synaptopodin family of proteins consists of at least 3 members: synaptopodin, the synaptopodin 2 proteins, and the synaptopodin 2-like proteins. Each family member has at least 3 isoforms that are produced by alternative splicing. Synaptopodin family members are basic proteins that are rich in proline and have little regular 2° or 3° structure at physiological temperature, pH and ionic strength. Like other natively unfolded proteins, synaptopodin family members have multiple binding partners including actin and other actin-binding proteins. Several members of the synaptopodin family have been shown to stimulate actin polymerization and to bundle actin filaments either on their own or in collaboration with other proteins. Synaptopodin 2 has been shown to accelerate nucleation of actin filament formation and to induce actin bundling. The actin polymerization activity is inhibited by Ca-calmodulin. Synaptopodin 2 proteins are localized in Z-bands of striated and heart muscle and dense bodies of smooth muscle cells. Depending on the developmental status and stress, at least one member of the synaptopodin family can occupy nuclei of some cells. Members of the synaptopodin 2 subfamily have been implicated in cancers.
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http://dx.doi.org/10.1007/s12551-010-0040-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418383PMC
December 2010

Phosphorylation of caldesmon at sites between residues 627 and 642 attenuates inhibitory activity and contributes to a reduction in Ca2+-calmodulin affinity.

Biophys J 2010 Sep;99(6):1861-8

Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina, USA.

Caldesmon is an actin- and myosin-binding protein found in smooth muscle that inhibits actin activation of myosin ATPase activity. The activity of caldesmon is controlled by phosphorylation and by binding to Ca(2+)-calmodulin. We investigated the effects of phosphorylation by p(21)-activated kinase 3 (PAK) and calmodulin on the 22 kDa C-terminal fragment of caldesmon (CaD22). We substituted the major PAK sites, Ser-672 and Ser-702, with either alanine or aspartic acid to mimic nonphosphorylated and constitutively phosphorylated states of caldesmon, respectively. The aspartic acid mutation of CaD22 weakened Ca(2+)-calmodulin binding but had no effect on inhibition of ATPase activity. Phosphorylation of the aspartic acid mutant with PAK resulted in the slow phosphorylation of Thr-627, Ser-631, Ser-635, and Ser-642. Phosphorylation at these sites weakened Ca(2+)-calmodulin binding further and reduced the inhibitory activity of CaD22 in the absence of Ca(2+)-calmodulin. Phosphorylation of these sites of the alanine mutant of CaD22 had no effect on Ca(2+)-calmodulin binding but did reduce inhibition of ATPase activity. Thus, the region between residues 627 and 642 may contribute to the overall regulation of caldesmon's activity.
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http://dx.doi.org/10.1016/j.bpj.2010.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2940999PMC
September 2010

Molecular dynamics studies on troponin (TnI-TnT-TnC) complexes: insight into the regulation of muscle contraction.

J Biomol Struct Dyn 2010 Oct;28(2):159-74

Department of Chemistry, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA.

Mutations of any subunit of the troponin complex may lead to serious disorders. Rational approaches to managing these disorders require knowledge of the complex interactions among the three subunits that are required for proper function. Molecular dynamics (MD) simulations were performed for both skeletal (sTn) and cardiac (cTn) troponin. The interactions and correlated motions among the three components of the troponin complex were analyzed using both Molecular Mechanics-Generalized Born Surface Area (MMGBSA) and cross-correlation techniques. The TnTH2 helix was strongly positively correlated with the two long helices of TnI. The C domain of TnC was positively correlated with TnI and TnT. The N domain of TnC was negatively correlated with TnI and TnT in cTn, but not in sTn. The two C-domain calcium-binding sites of TnC were dynamically correlated. The two regulatory N-domain calcium-binding sites of TnC were dynamically correlated, even though the calcium-binding site I is dysfunctional. The strong interaction residue pairs and the strong dynamically correlated residues pairs among the three components of troponin complexes were identified. These correlated motions are consistent with the idea that there is a high degree of cooperativity among the components of the regulatory complex in response to Ca(2+) and other effectors. This approach may give insight into the mechanism by which mutations of troponin cause disease. It is interesting that some observed disease causing mutations fall within regions of troponin that are strongly correlated or interacted.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807689PMC
http://dx.doi.org/10.1080/07391102.2010.10507350DOI Listing
October 2010

Kinetics of regulated actin transitions measured by probes on tropomyosin.

Biophys J 2010 Jun;98(11):2601-9

Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, USA.

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.
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http://dx.doi.org/10.1016/j.bpj.2010.02.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877322PMC
June 2010

Some cardiomyopathy-causing troponin I mutations stabilize a functional intermediate actin state.

Biophys J 2009 Mar;96(6):2237-44

Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, USA.

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.
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http://dx.doi.org/10.1016/j.bpj.2008.12.3909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2717271PMC
March 2009

Localization of the actin-binding protein fesselin in chicken smooth muscle.

Histochem Cell Biol 2009 Feb 27;131(2):191-6. Epub 2008 Sep 27.

Department of Anatomy and Cell Biology, Brody School of Medicine at East Carolina University, Greenville, NC, USA.

This report compares cellular localization of fesselin in chicken smooth, skeletal and cardiac muscle tissues using affinity purified polyclonal fesselin antibodies. Western blot analyses revealed large amounts of fesselin in gizzard smooth muscle with lower amounts in skeletal and cardiac muscle. In gizzard, fesselin was detected by immunofluorescence as discrete cytoplasmic structures. Fesselin did not co-localize with talin, vinculin or caveolin indicating that fesselin is not associated with dense plaques or caveolar regions of the cell membrane. Immunoelectron microscopy established localization of fesselin within dense bodies. Since dense bodies function as anchorage points for actin and desmin in smooth muscle cells, fesselin may be involved in establishing cytoskeletal structure in this tissue. In skeletal muscle, fesselin was associated with desmin in regularly spaced bands distributed along the length of muscle fibers suggesting localization to the Z-line. Infrequently, this banding pattern was observed in heart tissue as well. Localization at the Z-line of skeletal and cardiac muscle suggests a role in contraction of these tissues.
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http://dx.doi.org/10.1007/s00418-008-0508-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832296PMC
February 2009

In vitro characterization of native mammalian smooth-muscle protein synaptopodin 2.

Biosci Rep 2008 Aug;28(4):195-203

Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27834, USA.

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 Ca(2+)-calmodulin, alpha-actinin and smooth-muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca(2+)-calmodulin-dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of alpha-actinin, to polymerize actin in a Ca(2+)-calmodulin-dependent manner, or to bind to Ca(2+)-calmodulin or myosin. These properties are consistent with the proposed function of synaptopodin 2 in organizing the cytoskeleton.
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http://dx.doi.org/10.1042/BSR20080079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3763723PMC
August 2008