Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T.

Hinkle, Ashley; Goranson, Angela; Butters, Carol A.; Tobacman, Larry S.

doi:10.1016/s0021-9258(19)51681-7

Cited by 5 publications

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“…The calculated R S from these data was 21 ± 2 Å. 20,22,23 It is important to note that if we use the proposed linear relationship between the molecular weight and σ of the protein, 31 then the apparent molecular weight should be similar to that of chymotrypsin (eluting at 12.33 ± 0.06 mL) (second arrow from the right in Figure 3A of the Supporting Information). We did not use this relationship, instead of that between R S and σ (see Experimental Procedures), because in our column (Superdex 75 HR10, GE Healthcare) the linear relationship with the molecular weight and σ is worse than that with R S (regression coefficient of 0.95 vs 0.99).…”

Section: ■ Resultsmentioning

confidence: 99%

“…We attempted to fit the sigmoidal-like behavior of the elution volume of RDTyrH (Figure 2C, inset) to the Ackers' equations 19,21,22 assuming a monomer−dimer equilibrium:…”

Section: ■ Resultsmentioning

confidence: 99%

“…The standards used in column calibration and their corresponding R S values were as follows: ribonuclease A (16.4 Å), chymotrypsinogen (20.9 Å), ovalbumin (30.5 Å), and bovine serum albumin (35.5 Å). 22 Elution volumes of these were acquired only under native conditions: pH 7.0 (50 mM phosphate buffer) with 150 mM NaCl. Protein concentrations for the standards were in the range of 10−20 μM.…”

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confidence: 99%

“…The weight-average partition coefficients (σ) of protein standards and RD species were calculated with the equation σ = (V e − V o )/V i . The σ values were transformed by using the inverse error function complement [erfc −1 (σ)], which yields 21,22 where a and b are constants. The inverse error function complement is 23…”

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confidence: 99%

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The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability

et al. 2016

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Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine, the first step in the synthesis of catecholamine neurotransmitters. The protein contains a 159-residue regulatory domain (RD) at its N-terminus that forms dimers in solution; the N-terminal region of RDTyrH (residues 1-71) is absent in the solution structure of the domain. We have characterized the conformational stability of two species of RDTyrH (one containing the N-terminal region and another lacking the first 64 residues) to clarify how that N-terminal region modulates the conformational stability of RD. Under the conditions used in this study, the RD species lacking the first 64 residues is a monomer at pH 7.0, with a small conformational stability at 25 °C (4.7 ± 0.8 kcal mol(-1)). On the other hand, the entire RDTyrH is dimeric at physiological pH, with an estimated dissociation constant of 1.6 μM, as determined by zonal gel filtration chromatography; dimer dissociation was spectroscopically silent to circular dichroism but not to fluoresecence. Both RD species were disordered below physiological pH, but the acquisition of secondary native-like structure occurs at pHs lower than those measured for the attainment of tertiary native- and compactness-like arrangements.

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Section: ■ Resultsmentioning

confidence: 99%

“…We attempted to fit the sigmoidal-like behavior of the elution volume of RDTyrH (Figure 2C, inset) to the Ackers' equations 19,21,22 assuming a monomer−dimer equilibrium:…”

Section: ■ Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability

et al. 2016

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“…The largest mutation density within TnT, 15.6%, was in the TnT 87–150 helix that anchors troponin on the thin filament ( White et al, 1987 ; Hinkle et al, 1999 ; Gangadharan et al, 2017 ; Palm et al, 2001 ; Jin and Chong, 2010 ) and overlies the end-to-end connections between adjacent tropomyosins ( Yamada et al, 2020 ; White et al, 1987 ; Pavadai et al, 2020b ; Murakami et al, 2008 ). Several HCM- or DCM-causing causing mutations have been studied in great detail after being discovered in this region and in the evolutionarily conserved preceding few residues in the TnT sequence ( Watkins et al, 1995b ; Tardiff, 2011 ; Mirza et al, 2005 ; Gollapudi et al, 2015 ; Manning et al, 2012 ; McConnell et al, 2017 ).…”

Section: Resultsmentioning

confidence: 99%

Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

Tobacman

Cammarato

2021

Journal of General Physiology

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Reversible Ca2+ binding to troponin is the primary on-off switch of the contractile apparatus of striated muscles, including the heart. Dominant missense mutations in human cardiac troponin genes are among the causes of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy. Structural understanding of troponin action has recently advanced considerably via electron microscopy and molecular dynamics studies of the thin filament. As a result, it is now possible to examine cardiomyopathy-inducing troponin mutations in thin-filament structural context, and from that to seek new insight into pathogenesis and into the troponin regulatory mechanism. We compiled from consortium reports a representative set of troponin mutation sites whose pathogenicity was determined using standardized clinical genetics criteria. Another set of sites, apparently tolerant of amino acid substitutions, was compiled from the gnomAD v2 database. Pathogenic substitutions occurred predominantly in the areas of troponin that contact actin or tropomyosin, including, but not limited to, two regions of newly proposed structure and long-known implication in cardiomyopathy: the C-terminal third of troponin I and a part of the troponin T N terminus. The pathogenic mutations were located in troponin regions that prevent contraction under low Ca2+ concentration conditions. These regions contribute to Ca2+-regulated steric hindrance of myosin by the combined effects of troponin and tropomyosin. Loss-of-function mutations within these parts of troponin result in loss of inhibition, consistent with the hypercontractile phenotype characteristic of HCM. Notably, pathogenic mutations are absent in our dataset from the Ca2+-binding, activation-producing troponin C (TnC) N-lobe, which controls contraction by a multi-faceted mechanism. Apparently benign mutations are also diminished in the TnC N-lobe, suggesting mutations are poorly tolerated in that critical domain.

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Selective Deletion of the NH₂-Terminal Variable Region of Cardiac Troponin T in Ischemia Reperfusion by Myofibril-Associated μ-Calpain Cleavage

2006

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The structure of the NH2-terminal region of troponin T (TnT) is hypervariable among the muscle type-specific isoforms and is also regulated by alternative RNA splicing. This region does not contain binding sites for other thin filament proteins, but alteration of its structure affects the Ca2+ regulation of muscle contraction. Here we report a truncated cardiac TnT produced during myocardial ischemia reperfusion. Amino acid sequencing and protein fragment reconstruction determined that it is generated by a posttranslational modification selectively removing the NH2-terminal variable region and preserving the conserved core structure of TnT. Triton X-100 extraction of cardiac muscle fibers promoted production of the NH2-terminal truncated cardiac TnT (cTnT-ND), indicating a myofibril-associated proteolytic activity. Mu-calpain is a myofibril-associated protease and is known to degrade TnT. Supporting a role of mu-calpain in producing cTnT-ND in myocardial ischemia reperfusion, calpain inhibitors decreased the level of cTnT-ND in Triton-extracted myofibrils. Mu-calpain treatment of the cardiac myofibril and troponin complex specifically reproduced cTnT-ND. In contrast, mu-calpain treatment of isolated cardiac TnT resulted in nonspecific degradation, suggesting that this structural modification is relevant to physiological structures of the myofilament. Triton X-100 treatment of transgenic mouse cardiac myofibrils overexpressing fast skeletal muscle TnT produced similar NH2-terminal truncations of the endogenous and exogenous TnT, despite different amino acid sequences at the cleavage site. With the functional consequences of removing the NH2-terminal variable region of TnT, the mu-calpain-mediated proteolytic modification of TnT may act as an acute mechanism to adjust muscle contractility under stress conditions.

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Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T.

Cited by 5 publications

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The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability

The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability

Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

Selective Deletion of the NH₂-Terminal Variable Region of Cardiac Troponin T in Ischemia Reperfusion by Myofibril-Associated μ-Calpain Cleavage

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Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T.

Cited by 5 publications

References 0 publications

The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability

The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability

Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

Selective Deletion of the NH2-Terminal Variable Region of Cardiac Troponin T in Ischemia Reperfusion by Myofibril-Associated μ-Calpain Cleavage

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Selective Deletion of the NH₂-Terminal Variable Region of Cardiac Troponin T in Ischemia Reperfusion by Myofibril-Associated μ-Calpain Cleavage