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Fernando C Reinach - One of the best experts on this subject based on the ideXlab platform.

  • the calcium induced switch in the Troponin Complex probed by fluorescent mutants of Troponin i
    FEBS Journal, 2003
    Co-Authors: Deodoro Camargo Silva Goncalves De Oliveira, Fernando C Reinach
    Abstract:

    The Ca2+-induced transition in the Troponin Complex (Tn) regulates vertebrate striated muscle contraction. Tn was reconstituted with recombinant forms of Troponin I (TnI) containing a single intrinsic 5-hydroxytryptophan (5HW). Fluorescence analysis of these mutants of TnI demonstrate that the regions in TnI that respond to Ca2+ binding to the regulatory N-domain of TnC are the inhibitory region (residues 96-116) and a neighboring region that includes position 121. Our data confirms the role of TnI as a modulator of the Ca2+ affinity of TnC; we show that point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC. We also discuss the possibility that the regulatory sites in the N-terminal domain of TnC might be the high affinity Ca2+-binding sites in the Troponin Complex.

  • structural interactions responsible for the assembly of the Troponin Complex on the muscle thin filament
    Cell Structure and Function, 1997
    Co-Authors: Fernando C Reinach, Chuck S Farah, P B Monteiro, Bettina Malnic
    Abstract:

    Skeletal muscle contraction is regulated by a Complex of five polypeptides which are stably associated with the actin filament. This Complex consists of two proteins: Troponin with three subunits (TnC; TnI and TnT) and tropomyosin (a dimer of two chains). Using deletion mutants of TnC, TnI and TnT we determined that each of these polypeptides can be divided into at least two domains. One domain is responsible for the regulatory properties of the protein. Its interaction with the other components of the system change upon calcium binding to TnC. A second domain present in each of these proteins is responsible for the stable association of the Complex to the actin filament. The interactions among this second set of domains is not influenced by calcium binding to TnC. The structural interactions are: 1) interactions between the C-domain of TnC with the N-domain of TnI; 2) interactions of the N-domain of TnI with the C-terminal domain of TnT and 3) interactions between the N-domain of TnT (T1) and actin/tropomyosin.

  • the Troponin Complex and regulation of muscle contraction
    The FASEB Journal, 1995
    Co-Authors: Chuck S Farah, Fernando C Reinach
    Abstract:

    In a wide variety of cellular settings, from organelle transport to muscle contraction, Ca2+ binding to members of the EF hand family of proteins controls the interaction between actin and different myosins that are responsible for generating movement. In vertebrate skeletal and cardiac muscle the Ca(2+)-binding protein Troponin C (TnC) is one subunit of the ternary Troponin Complex which, through its association with actin and tropomyosin on the thin filament, inhibits the actomyosin interaction at submicromolar Ca2+ concentrations and stimulates the interaction at micromolar Ca2+ concentrations. Because TnC does not interact directly with actin or tropomyosin, the Ca(2+)-binding signal must be transmitted to the thin filament via the other two Troponin subunits: Troponin I (TnI), the inhibitory subunit, and Troponin T (TnT), the tropomyosin-binding subunit. Thus, the Troponin Complex is a Ca(2+)-sensitive molecular switch and the structures of and interactions between its components have been of great i...

  • assembly of functional skeletal muscle Troponin Complex in escherichia coli
    FEBS Journal, 1994
    Co-Authors: Bettina Malnic, Fernando C Reinach
    Abstract:

    The production of multi-subunit proteins of eukaryotic origin in Escherichia coli usually relies on the different subunits being expressed individually and the protein being reassembled in vitro. Here we describe the construction and characterization of plasmids capable of coexpressing the three subunits of chicken skeletal muscle Troponin Complex in E. coli. We demonstrate that the Troponin subunits assembled in the cytoplasm of E. coli cell are fully functional. The Troponin Complex was purified to homogeneity in high yields. When reconstituted into actin filaments, the Complex assembled in vivo was capable of regulating the myosin ATPase with a calcium dependence that was identical to the Complex reconstituted in vitro. These results demonstrate that the coexpression of the subunits of a protein Complex can prevent the accumulation of denatured proteins in inclusion granules.

Tomoyoshi Kobayashi - One of the best experts on this subject based on the ideXlab platform.

  • sites of intra and intermolecular cross linking of the n terminal extension of Troponin i in human cardiac whole Troponin Complex
    Journal of Biological Chemistry, 2009
    Co-Authors: Chad M Warren, Tomoyoshi Kobayashi, John R. Solaro
    Abstract:

    Our previous studies (Howarth, J. W., Meller, J., Solaro, R. J., Trewhella, J., and Rosevear, P. R. (2007) J. Mol. Biol. 373, 706–722) of the unique N-terminal region of human cardiac Troponin I (hcTnI), predicted a possible intramolecular interaction near the basic inhibitory peptide. To explore this possibility, we generated single cysteine mutants (hcTnI-S5C and hcTnI-I19C), which were labeled with the hetero-bifunctional cross-linker benzophenone-4-maleimide. The labeled hcTnI was reconstituted to whole Troponin and exposed to UV light to form cross-linked proteins. Reversed-phase high-performance liquid chromatography and SDS-PAGE indicated intra- and intermolecular cross-linking with hcTnC and hcTnT. Moreover, using tandem mass spectrometry and Edman sequencing, specific intramolecular sites of interaction were determined at position Met-154 (I19C mutant) and Met-155 (S5C mutant) of hcTnI and intermolecular interactions at positions Met-47 and Met-80 of hcTnC in all conditions. Even though specific intermolecular cross-linked sites did not differ, the relative abundance of cross-linking was altered. We also measured the Ca2+-dependent ATPase rate of reconstituted thin filament-myosin-S1 preparation regulated by either cross-linked or non-labeled Troponin. Ca2+ regulation of the ATPase rate was lost when the Cys-5 hcTnI mutant was cross-linked in the absence of Ca2+, but only partially inhibited with Cys-19 cross-linking in either the presence or absence of Ca2+. This result indicates different functional effects of cross-linking to Met-154 and Met-155, which are located on different sides of the hcTnI switch peptide. Our data provide novel evidence identifying interactions of the hcTnI-N terminus with specific intra- and intermolecular sites.

  • the Troponin c g159d mutation blunts myofilament desensitization induced by Troponin i ser23 24 phosphorylation
    Circulation Research, 2007
    Co-Authors: Brandon J. Biesiadecki, Tomoyoshi Kobayashi, John R. Solaro, John S Walker, Pieter P De Tombe
    Abstract:

    Striated muscle contraction is regulated by the binding of Ca(2+) to the N-terminal regulatory lobe of the cardiac Troponin C (cTnC) subunit in the Troponin Complex. In the heart, beta-adrenergic stimulation induces protein kinase A phosphorylation of cardiac Troponin I (cTnI) at Ser23/24 to alter the interaction of cTnI with cTnC in the Troponin Complex and is critical to the regulation of cardiac contractility. We investigated the effect of the dilated cardiomyopathy linked cTnC Gly159 to Asp (cTnC-G159D) mutation on the development of Ca(2+)-dependent tension and ATPase rate in whole Troponin-exchanged skinned rat trabeculae. Even though this mutation is located in the C-terminal lobe of cTnC, the G159D mutation was demonstrated to depress ATPase activation and filament sliding in vitro. The effects of this mutation within the cardiac myofilament are unknown. Our results demonstrate that the cTnC-G159D mutation by itself does not alter the myofilament response to Ca(2+) in the cardiac muscle lattice. However, in the presence of cTnI phosphorylated at Ser23/24, which reduced Ca(2+) sensitivity and enhanced cross-bridge cycling in controls, cTnC-G159D specifically blunted the phosphorylation induced decrease in Ca(2+)-sensitive tension development without altering cross-bridge cycling. Measurements in purified Troponin confirmed that this cTnC-G159D blunting of myofilament desensitization results from altered Ca(2+)-binding to cTnC. Our results provide novel evidence that modification of the cTnC-cTnI interaction has distinct effects on Troponin Ca(2+)-binding and cross-bridge kinetics to suggest a novel role for thin filament mutations in the modulation of myofilament function through beta-adrenergic signaling as well as the development of cardiomyopathy.

  • effects of thin and thick filament proteins on calcium binding and exchange with cardiac Troponin c
    Biophysical Journal, 2007
    Co-Authors: Jonathan P. Davis, Tomoyoshi Kobayashi, John R. Solaro, Catalina Norman, Darl R Swartz, Svetlana B. Tikunova
    Abstract:

    Understanding the effects of thin and thick filament proteins on the kinetics of Ca2+ exchange with cardiac Troponin C is essential to elucidating the Ca2+-dependent mechanisms controlling cardiac muscle contraction and relaxation. Unlike labeling of the endogenous Cys-84, labeling of cardiac Troponin C at a novel engineered Cys-53 with 2-(4′-iodoacetamidoanilo)napthalene-6-sulfonic acid allowed us to accurately measure the rate of calcium dissociation from the regulatory domain of Troponin C upon incorporation into the Troponin Complex. Neither tropomyosin nor actin alone affected the Ca2+ binding properties of the Troponin Complex. However, addition of actin-tropomyosin to the Troponin Complex decreased the Ca2+ sensitivity (∼7.4-fold) and accelerated the rate of Ca2+ dissociation from the regulatory domain of Troponin C (∼2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin filaments (actin-tropomyosin-Troponin) increased the Ca2+ sensitivity (∼6.2-fold) and decreased the rate of Ca2+ dissociation from the regulatory domain of Troponin C (∼8.1-fold), which was completely reversed by ATP. Consistent with physiological data, replacement of cardiac Troponin I with slow skeletal Troponin I led to higher Ca2+ sensitivities and slower Ca2+ dissociation rates from Troponin C in all the systems studied. Thus, both thin and thick filament proteins influence the ability of cardiac Troponin C to sense and respond to Ca2+. These results imply that both cross-bridge kinetics and Ca2+ dissociation from Troponin C work together to modulate the rate of cardiac muscle relaxation.

  • identification of a functionally critical protein kinase c phosphorylation residue of cardiac Troponin t
    Journal of Biological Chemistry, 2003
    Co-Authors: Marius P. Sumandea, Tomoyoshi Kobayashi, Pieter P De Tombe, Glen W Pyle, John R. Solaro
    Abstract:

    Abstract Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous Troponin was replaced with a recombinant Troponin Complex containing either cTnT phosphorylated by PKC-α or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-α or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.

  • structural and functional domains of the Troponin Complex revealed by limited digestion
    FEBS Journal, 1997
    Co-Authors: Tomoyoshi Kobayashi, Soichi Takeda, Hisaaki Taniguchi, Hiroshi Hayashi, Yuichiro Maeda
    Abstract:

    Troponin (Tn), consisting of three subunits, TnT, TnC, and TnI, plays a crucial role in the calcium-dependent regulation of vertebrate striated muscle contraction. In the present study, we have applied limited proteolysis to the Tn Complex in order to study domain structures and to detect conformational differences of Tn under different conditions. We found that both TnT and TnI were susceptible to chymotryptic digestion: while TnT was cleaved into TnT-(1-158)-peptide and TnT-(159-259)-peptide irrespective of Ca2+ concentration, the cleavage sites of TnI were dependent on the Ca2+ occupancy of TnC. In addition, we characterized the effects of depletion of the C-terminal part of TnI on acto-S1 ATPase activity. The TnT-(159-259)-peptide-TnC-TnICa-frag Complex [TnICa-frag = (TnI-(1-134 and 1-140)-peptide], which was produced in the presence of CaCl2 and MgCl2, retains both the activating and inhibitory capabilities of whole Tn on the acto-S1 ATPase activity, while TnT-(159-259)-peptide-TnC-TnIMg-frag Complex [TnIMg-frag = (TnI-(1-116)-peptide], which was obtained in the presence of MgCl2 and EGTA, lost its ability to activate acto-S1 ATPase activity. Our results indicate that residues 117-134 or 117-140 of TnI undergo structural changes upon Ca(2+)-binding to the regulatory sites of TnC and are necessary for the Ca(2+)-dependent inhibitory action of the Tn Complex on acto-S1 ATPase activity. We also showed that residues 135-181 or 141-181 of TnI are involved in the interaction of Tn with the tropomyosin-actin filament.

Paul M G Curmi - One of the best experts on this subject based on the ideXlab platform.

  • ca2 induced pre nmr changes in the Troponin Complex reveal the possessive nature of the cardiac isoform for its regulatory switch
    PLOS ONE, 2014
    Co-Authors: Nicole M Cordina, Piotr G Fajer, Paul M G Curmi, Chu K Liew, Phani R Potluri, Timothy M Logan, Joel P Mackay, Louise J Brown
    Abstract:

    The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca2+) sensor Troponin Complex (Tn), is a Complex process which is yet to be fully resolved at the molecular level. Our understanding of how the binding of Ca2+ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca2+-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE)-NMR to obtain a description of the Ca2+-free state of cardiac Tn by describing the movement of key regions of the Troponin I (cTnI) subunit upon the release of Ca2+ from Troponin C (cTnC). Site-directed spin-labeling was used to position paramagnetic spin labels in cTnI and the changes in the interaction between cTnI and cTnC subunits were then mapped by PRE-NMR. The functionally important regions of cTnI targeted in this study included the cTnC-binding N-region (cTnI57), the inhibitory region (cTnI143), and two sites on the regulatory switch region (cTnI151 and cTnI159). Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 A) away from the hydrophobic N-lobe of Troponin C (cTnC) upon the removal of Ca2+. Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca2+ but remains in close vicinity to cTnC. We speculate that the close proximity of TnI to TnC in the cardiac Complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.

  • solution structure of the chicken skeletal muscle Troponin Complex via small angle neutron and x ray scattering
    Journal of Molecular Biology, 2005
    Co-Authors: William A King, Robert A. Mendelson, Deborah B. Stone, Peter A Timmins, Theyencheri Narayanan, Alex A M Von Brasch, Paul M G Curmi
    Abstract:

    Troponin is a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle by participating in a series of conformational events within the actin-based thin filament. Troponin is a heterotrimeric Complex consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT). Ternary Troponin Complexes have been produced by assembling recombinant chicken skeletal muscle TnC, TnI and the C-terminal portion of TnT known as TnT2. A full set of small-angle neutron scattering data has been collected from TnC-TnI-TnT2 ternary Complexes, in which all possible combinations of the subunits have been deuterated, in both the +Ca2+ and -Ca2+ states. Small-angle X-ray scattering data were also collected from the same Troponin TnC-TnI-TnT2 Complex. Guinier analysis shows that the Complex is monomeric in solution and that there is a large change in the radius of gyration of TnI when it goes from the +Ca2+ to the -Ca2+ state. Starting with a model based on the human cardiac Troponin crystal structure, a rigid-body Monte Carlo optimization procedure was used to yield models of chicken skeletal muscle Troponin, in solution, in the presence and in the absence of regulatory calcium. The optimization was carried out simultaneously against all of the scattering data sets. The optimized models show significant differences when compared to the cardiac Troponin crystal structure in the +Ca2+ state and provide a structural model for the switch between +Ca2+ and -Ca2+ states. A key feature is that TnC adopts a dumbbell conformation in both the +Ca2+ and -Ca2+ states. More importantly, the data for the -Ca2+ state suggest a long extension of the Troponin IT arm, consisting mainly of TnI. Thus, the Troponin Complex undergoes a large structural change triggered by Ca2+ binding.

Brian D. Sykes - One of the best experts on this subject based on the ideXlab platform.

  • The calcium sensitizer drug MCI-154 binds the structural C-terminal domain of cardiac Troponin C
    Elsevier, 2018
    Co-Authors: Shorena Gelozia, Brian D. Sykes, Gaddafi I. Danmaliki, Yurong Wen, Philip B. Liu, Joanne M. Lemieux, Frederick G. West, Peter M. Hwang
    Abstract:

    The compound MCI-154 was previously shown to increase the calcium sensitivity of cardiac muscle contraction. Using solution NMR spectroscopy, we demonstrate that MCI-154 interacts with the calcium-sensing subunit of the cardiac Troponin Complex, cardiac Troponin C (cTnC). Surprisingly, however, it binds only to the structural C-terminal domain of cTnC (cCTnC), and not to the regulatory N-terminal domain (cNTnC) that determines the calcium sensitivity of cardiac muscle.Physiologically, cTnC is always bound to cardiac Troponin I (cTnI), so we examined its interaction with MCI-154 in the presence of two soluble constructs, cTnI1–77 and cTnI135–209, which contain all of the segments of cTnI known to interact with cTnC. Neither the cTnC-cTnI1–77 Complex nor the cTnC-cTnI135–209 Complex binds to MCI-154. Since residues 39–60 of cTnI are known to bind tightly to the cCTnC domain to form a structured core that is invariant throughout the cardiac cycle, we conclude that MCI-154 does not bind to cTnC when it is part of the intact cardiac Troponin Complex. Thus, MCI-154 likely exerts its calcium sensitizing effect by interacting with a target other than cardiac Troponin. Keywords: Solution NMR spectroscopy, Calcium sensitizer, Drug binding, Protein-protein interactio

  • targeting the sarcomere to correct muscle function
    Nature Reviews Drug Discovery, 2015
    Co-Authors: Peter M. Hwang, Brian D. Sykes
    Abstract:

    Various human diseases can disrupt the balance between muscle contraction and relaxation. Sarcomeric modulators can be used to readjust this balance either indirectly by intervening in signalling pathways or directly through interaction with the muscle proteins that control contraction. Such agents represent a novel approach to treating any condition in which striated muscle function is compromised, including heart failure, cardiomyopathies, skeletal myopathies and a wide range of neuromuscular conditions. Here, we review agents that modulate the mechanical function of the sarcomere, focusing on emerging compounds that target myosin or the Troponin Complex.

  • Dynamics of a Troponin Chimera Reproduce the Effects of Calcium on the Troponin Complex
    Biophysical Journal, 2012
    Co-Authors: Olivier Julien, Tharin M A Blumenschein, Claire N. Allen, Pascal Mercier, Carlos H.i. Ramos, Brian D. Sykes
    Abstract:

    In striated muscle, contraction is regulated in a Ca2+-dependent manner by the three subunits of the Troponin Complex. Troponin I (TnI) inhibits actomyosin ATPase in the absence of calcium; Ca2+ binding to Troponin C (TnC) causes conformational changes that alter the interaction between TnI and TnC, removing the inhibition. These conformational changes are transmitted to the rest of the thin filament through interactions with Troponin T (TnT). Nuclear magnetic resonance (NMR) studies of the core skeletal Troponin Complex (52 kDa) showed Ca2+-dependent changes in relaxation parameters of the regulatory region of TnC (residues 1-91) (Blumenschein et al., J. Biol. Chem. 280, 21924), and that the last 50 residues of TnI are disordered irrespective of the presence of calcium (Blumenschein et al., Biophys. J. 90, 2436). This disorder is postulated to be essential for muscle regulation (Hoffmann et al., J. Mol. Biol. 361, 625). Due to the size of the Troponin Complex, it was not possible to observe the remaining residues of the regulatory region of TnI (98-182). A chimeric polypeptide, containing the regulatory regions of TnI and TnC connected by a short linker (GGAGG), is capable of regulating actomyosin ATPase (Tiroli et al., FEBS Journal 272, 779), and at 20 kDa, provides a better target for NMR studies and the ability to visualise the residues so far unobserved. NMR relaxation measurements were used to study the dynamics of this Troponin chimera in the presence and absence of calcium, and when the difference in molecular weight is taken into account, the relaxation parameters reproduce perfectly the results observed for the whole Complex, in the presence and absence of calcium, both for the TnC and TnI regions previously observed.

  • a structural and functional perspective into the mechanism of ca2 sensitizers that target the cardiac Troponin Complex
    Journal of Molecular and Cellular Cardiology, 2010
    Co-Authors: Ian M Robertson, Yinbiao Sun, Brian D. Sykes
    Abstract:

    Abstract The Ca2+ dependant interaction between Troponin I (cTnI) and Troponin C (cTnC) triggers contraction in heart muscle. Heart failure is characterized by a decrease in cardiac output, and compounds that increase the sensitivity of cardiac muscle to Ca2+ have therapeutic potential. The Ca2+-sensitizer, levosimendan, targets cTnC; however, detailed understanding of its mechanism has been obscured by its instability. In order to understand how this class of positive inotropes function, we investigated the mode of action of two fluorine containing novel analogs of levosimendan; 2′,4′-difluoro(1,1′-biphenyl)-4-yloxy acetic acid (dfbp-o) and 2′,4′-difluoro(1,1′-biphenyl)-4-yl acetic acid (dfbp). The affinities of dfbp and dfbp-o for the regulatory domain of cTnC were measured in the absence and presence of cTnI by NMR spectroscopy, and dfbp-o was found to bind more strongly than dfbp. Dfbp-o also increased the affinity of cTnI for cTnC. Dfbp-o increased the Ca2+-sensitivity of demembranated cardiac trabeculae in a manner similar to levosimendan. The high resolution NMR solution structure of the cTnC–cTnI–dfbp-o ternary Complex showed that dfbp-o bound at the hydrophobic interface formed by cTnC and cTnI making critical interactions with residues such as Arg147 of cTnI. In the absence of cTnI, docking localized dfbp-o to the same position in the hydrophobic groove of cTnC. The structural and functional data reveal that the levosimendan class of Ca2+-sensitizers work by binding to the regulatory domain of cTnC and stabilizing the pivotal cTnC–cTnI regulatory unit via a network of hydrophobic and electrostatic interactions, in contrast to the destabilizing effects of antagonists such as W7 at the same interface.

  • dynamics of the c terminal region of tni in the Troponin Complex in solution
    Biophysical Journal, 2006
    Co-Authors: Tharin M A Blumenschein, Robert A. Mendelson, Deborah B. Stone, Robert J Fletterick, Brian D. Sykes
    Abstract:

    The determination of crystal structures of the Troponin Complex (Takeda et al. 2003. Nature. 424:35–41; Vinogradova et al. 2005. Proc. Natl. Acad. Sci. USA. 102:5038–5043) has advanced knowledge of the regulation of muscle contraction at the molecular level. However, there are domains important for actin binding that are not visualized. We present evidence that the C-terminal region of Troponin I (TnI residues 135–182) is flexible in solution and has no stable secondary structure. We use NMR spectroscopy to observe the backbone dynamics of skeletal [2H, 13C, 15N]-TnI in the Troponin Complex in the presence of Ca2+ or EGTA/Mg2+. Residues in this region give stronger signals than the remainder of TnI, and chemical shift index values indicate little secondary structure, suggesting a very flexible region. This is confirmed by NMR relaxation measurements. Unlike TnC and other regions of TnI in the Complex, the C-terminal region of TnI is not affected by Ca2+ binding. Relaxation measurements and reduced spectral density analysis are consistent with the C-terminal region of TnI being a tethered domain connected to the rest of the Troponin Complex by a flexible linker, residues 137–146, followed by a collapsed region with at most nascent secondary structure.

John R. Solaro - One of the best experts on this subject based on the ideXlab platform.

  • implications of the Complex biology and micro environment of cardiac sarcomeres in the use of high affinity Troponin antibodies as serum biomarkers for cardiac disorders
    Journal of Molecular and Cellular Cardiology, 2020
    Co-Authors: Christopher R Solaro, John R. Solaro
    Abstract:

    Abstract Cardiac Troponin I (cTnI), the inhibitory-unit, and cardiac Troponin T (cTnT), the tropomyosin-binding unit together with the Ca-binding unit (cTnC) of the hetero-trimeric Troponin Complex signal activation of the sarcomeres of the adult cardiac myocyte. The unique structure and heart myocyte restricted expression of cTnI and cTnT led to their worldwide use as biomarkers for acute myocardial infarction (AMI) beginning more than 30 years ago. Over these years, high sensitivity antibodies (hs-cTnI and hs-cTnT) have been developed. Together with careful determination of history, physical examination, and EKG, determination of serum levels using hs-cTnI and hs-cTnT permits risk stratification of patients presenting in the Emergency Department (ED) with chest pain. With the ability to determine serum levels of these Troponins with high sensitivity came the question of whether such measurements may be of diagnostic and prognostic value in conditions beyond AMI. Moreover, the finding of elevated serum Troponins in physiological states such as exercise and pathological states where cardiac myocytes may be affected requires understanding of how Troponins may be released into the blood and whether such release may be benign. We consider these questions by relating membrane stability to the Complex biology of Troponin with emphasis on its sensitivity to the chemo-mechanical and micro-environment of the cardiac myocyte. We also consider the role determinations of serum Troponins play in the precise phenotyping in personalized and precision medicine approaches to promote cardiac health.

  • sites of intra and intermolecular cross linking of the n terminal extension of Troponin i in human cardiac whole Troponin Complex
    Journal of Biological Chemistry, 2009
    Co-Authors: Chad M Warren, Tomoyoshi Kobayashi, John R. Solaro
    Abstract:

    Our previous studies (Howarth, J. W., Meller, J., Solaro, R. J., Trewhella, J., and Rosevear, P. R. (2007) J. Mol. Biol. 373, 706–722) of the unique N-terminal region of human cardiac Troponin I (hcTnI), predicted a possible intramolecular interaction near the basic inhibitory peptide. To explore this possibility, we generated single cysteine mutants (hcTnI-S5C and hcTnI-I19C), which were labeled with the hetero-bifunctional cross-linker benzophenone-4-maleimide. The labeled hcTnI was reconstituted to whole Troponin and exposed to UV light to form cross-linked proteins. Reversed-phase high-performance liquid chromatography and SDS-PAGE indicated intra- and intermolecular cross-linking with hcTnC and hcTnT. Moreover, using tandem mass spectrometry and Edman sequencing, specific intramolecular sites of interaction were determined at position Met-154 (I19C mutant) and Met-155 (S5C mutant) of hcTnI and intermolecular interactions at positions Met-47 and Met-80 of hcTnC in all conditions. Even though specific intermolecular cross-linked sites did not differ, the relative abundance of cross-linking was altered. We also measured the Ca2+-dependent ATPase rate of reconstituted thin filament-myosin-S1 preparation regulated by either cross-linked or non-labeled Troponin. Ca2+ regulation of the ATPase rate was lost when the Cys-5 hcTnI mutant was cross-linked in the absence of Ca2+, but only partially inhibited with Cys-19 cross-linking in either the presence or absence of Ca2+. This result indicates different functional effects of cross-linking to Met-154 and Met-155, which are located on different sides of the hcTnI switch peptide. Our data provide novel evidence identifying interactions of the hcTnI-N terminus with specific intra- and intermolecular sites.

  • the Troponin c g159d mutation blunts myofilament desensitization induced by Troponin i ser23 24 phosphorylation
    Circulation Research, 2007
    Co-Authors: Brandon J. Biesiadecki, Tomoyoshi Kobayashi, John R. Solaro, John S Walker, Pieter P De Tombe
    Abstract:

    Striated muscle contraction is regulated by the binding of Ca(2+) to the N-terminal regulatory lobe of the cardiac Troponin C (cTnC) subunit in the Troponin Complex. In the heart, beta-adrenergic stimulation induces protein kinase A phosphorylation of cardiac Troponin I (cTnI) at Ser23/24 to alter the interaction of cTnI with cTnC in the Troponin Complex and is critical to the regulation of cardiac contractility. We investigated the effect of the dilated cardiomyopathy linked cTnC Gly159 to Asp (cTnC-G159D) mutation on the development of Ca(2+)-dependent tension and ATPase rate in whole Troponin-exchanged skinned rat trabeculae. Even though this mutation is located in the C-terminal lobe of cTnC, the G159D mutation was demonstrated to depress ATPase activation and filament sliding in vitro. The effects of this mutation within the cardiac myofilament are unknown. Our results demonstrate that the cTnC-G159D mutation by itself does not alter the myofilament response to Ca(2+) in the cardiac muscle lattice. However, in the presence of cTnI phosphorylated at Ser23/24, which reduced Ca(2+) sensitivity and enhanced cross-bridge cycling in controls, cTnC-G159D specifically blunted the phosphorylation induced decrease in Ca(2+)-sensitive tension development without altering cross-bridge cycling. Measurements in purified Troponin confirmed that this cTnC-G159D blunting of myofilament desensitization results from altered Ca(2+)-binding to cTnC. Our results provide novel evidence that modification of the cTnC-cTnI interaction has distinct effects on Troponin Ca(2+)-binding and cross-bridge kinetics to suggest a novel role for thin filament mutations in the modulation of myofilament function through beta-adrenergic signaling as well as the development of cardiomyopathy.

  • effects of thin and thick filament proteins on calcium binding and exchange with cardiac Troponin c
    Biophysical Journal, 2007
    Co-Authors: Jonathan P. Davis, Tomoyoshi Kobayashi, John R. Solaro, Catalina Norman, Darl R Swartz, Svetlana B. Tikunova
    Abstract:

    Understanding the effects of thin and thick filament proteins on the kinetics of Ca2+ exchange with cardiac Troponin C is essential to elucidating the Ca2+-dependent mechanisms controlling cardiac muscle contraction and relaxation. Unlike labeling of the endogenous Cys-84, labeling of cardiac Troponin C at a novel engineered Cys-53 with 2-(4′-iodoacetamidoanilo)napthalene-6-sulfonic acid allowed us to accurately measure the rate of calcium dissociation from the regulatory domain of Troponin C upon incorporation into the Troponin Complex. Neither tropomyosin nor actin alone affected the Ca2+ binding properties of the Troponin Complex. However, addition of actin-tropomyosin to the Troponin Complex decreased the Ca2+ sensitivity (∼7.4-fold) and accelerated the rate of Ca2+ dissociation from the regulatory domain of Troponin C (∼2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin filaments (actin-tropomyosin-Troponin) increased the Ca2+ sensitivity (∼6.2-fold) and decreased the rate of Ca2+ dissociation from the regulatory domain of Troponin C (∼8.1-fold), which was completely reversed by ATP. Consistent with physiological data, replacement of cardiac Troponin I with slow skeletal Troponin I led to higher Ca2+ sensitivities and slower Ca2+ dissociation rates from Troponin C in all the systems studied. Thus, both thin and thick filament proteins influence the ability of cardiac Troponin C to sense and respond to Ca2+. These results imply that both cross-bridge kinetics and Ca2+ dissociation from Troponin C work together to modulate the rate of cardiac muscle relaxation.

  • biology of the Troponin Complex in cardiac myocytes
    Progress in Cardiovascular Diseases, 2004
    Co-Authors: Michael S Parmacek, John R. Solaro
    Abstract:

    Troponin is the regulatory Complex of the myofibrillar thin filament that plays a critical role in regulating excitation-contraction coupling in the heart. Troponin is composed of three distinct gene products: Troponin C (cTnC), the 18-kD Ca(2+)-binding subunit; Troponin I (cTnI), the approximately 23-kD inhibitory subunit that prevents contraction in the absence of Ca2+ binding to cTnC; and Troponin T (cTnT), the approximately 35-kD subunit that attaches Troponin to tropomyosin (Tm) and to the myofibrillar thin filament. Over the past 45 years, extensive biochemical, biophysical, and structural studies have helped to elucidate the molecular basis of Troponin function and thin filament activation in the heart. At the onset of systole, Ca2+ binds to the N-terminal Ca2+ binding site of cTnC initiating a conformational change in cTnC, which catalyzes protein-protein associations activating the myofibrillar thin filament. Thin filament activation in turn facilitates crossbridge cycling, myofibrillar activation, and contraction of the heart. The intrinsic length-tension properties of cardiac myocytes as well as the Frank-Starling properties of the intact heart are mediated primarily through Ca(2+)-responsive thin filament activation. cTnC, cTnI, and cTnT are encoded by distinct single-copy genes in the human genome, each of which is expressed in a unique cardiac-restricted developmentally regulated fashion. Elucidation of the transcriptional programs that regulate Troponin transcription and gene expression has provided insights into the molecular mechanisms that regulate and coordinate cardiac myocyte differentiation and provided unanticipated insights into the pathogenesis of cardiac hypertrophy. Autosomal dominant mutations in cTnI and cTnT have been identified and are associated with familial hypertrophic and restrictive cardiomyopathies.