Solution Structure of the Chicken Skeletal Muscle Troponin Complex Via Small-angle Neutron and X-ray Scattering

King, W. A.; Stone, Deborah B.; Timmins, Peter; Narayanan, Theyencheri; Brasch, Alex A.M. von; Mendelson, Robert A.; Curmi, Paul M. G.

doi:10.1016/j.jmb.2004.10.090

Cited by 39 publications

(31 citation statements)

References 72 publications

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“…Substitution of cysteineless TnI for wild-type TnI in the Tn complex does not alter the Ca 2ϩ -dependent regulation of myosin ATPase activity (26). A uniformly deuterated deletion mutant of chicken skeletal muscle TnT (isoform TnT-3), corresponding to the T2 domain, was expressed and purified as described (25). 13 C, 15 N-and 2 H, 13 C, 15 N-labeled recombinant chicken skeletal TnC were expressed as described (27).…”

Section: Methodsmentioning

confidence: 99%

“…For [ 2 H, 13 C, 15 N]TnC, Escherichia coli cells conditioned to grow in medium containing D 2 O were used, and the medium used for expression contained D 2 O instead of H 2 O. The troponin complex containing these subunits was assembled and purified as described (25). The resulting complex was concentrated, dialyzed against 100 mM NH 4 HCO 3 , 0.1 mM DTT, and lyophilized.…”

Section: Methodsmentioning

confidence: 99%

“…The T1 domain of TnT was not included, because it causes aggregation of the complex (24,25), and it does not interact with TnC and TnI (2). We used this complex to measure backbone amide 15 N NMR relaxation times, which give information about the rotational tumbling and internal dynamics of proteins (20).…”

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confidence: 99%

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Calcium-dependent Changes in the Flexibility of the Regulatory Domain of Troponin C in the Troponin Complex

Blumenschein

Stone

Fletterick³

et al. 2005

Journal of Biological Chemistry

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With the recent advances in structure determination of the troponin complex, it becomes even more important to understand the dynamics of its components and how they are affected by the presence or absence of Ca 2؉ . We used NMR techniques to study the backbone dynamics of skeletal troponin C (TnC) in the complex. Transverse relaxation-optimized spectroscopy pulse sequences and deuteration of TnC were essential to assign most of the TnC residues in the complex. Backbone amide 15 N relaxation times were measured in the presence of Ca 2؉ or EGTA/Mg 2؉ . T 1 relaxation times could not be interpreted precisely, because for a molecule of this size, the longitudinal backbone amide 15 N relaxation rate due to chemical shift anisotropy and dipole-dipole interactions becomes too small, and other relaxation mechanisms become relevant. T 2 relaxation times were of the expected magnitude for a complex of this size, and most of the variation of T 2 times in the presence of Ca 2؉ could be explained by the anisotropy of the complex, suggesting a relatively rigid molecule. The only exception was EF-hand site III and helix F immediately after, which are more flexible than the rest of the molecule. In the presence of EGTA/Mg 2؉ , relaxation times for residues in the C-domain of TnC are very similar to values in the presence of Ca 2؉ , whereas the N-domain becomes more flexible. Taken together with the high flexibility of the linker between the two domains, we concluded that in the absence of Ca 2؉ , the N-domain of TnC moves independently from the rest of the complex.The troponin (Tn) 1 complex is responsible for the regulation of contraction in striated skeletal and cardiac muscles. Although many structural studies have been done of pairwise interactions between components of the complex (for reviews see Refs. 1-4), the details at an atomic level of how the complex works as a whole have not as yet been fully elucidated. Recent determination of the crystal structure of cardiac troponin (5) has revealed an apparently quite flexible complex. This has focused interest on the need for dynamics information on the troponin complex in order to properly interpret the structural data.The three components of Tn, troponins I, C, and T, are responsible for inhibiting muscle contraction in the absence of Ca 2ϩ , sensing the change in intracellular Ca 2ϩ levels, releasing the inhibition in the presence of Ca 2ϩ , and transmitting this information to the other components of muscle thin filaments, respectively (for a recent review see Ref.2). Troponin C (TnC) is a Ca 2ϩ -binding protein containing two domains connected by a linker. The linker is a straight ␣-helix in the crystal structure of isolated TnC (6 -8) but is flexible in solution (9). Each domain contains two EF-hand metal-binding sites, and the helices that flank those sites are named A to D in the N-domain and are named E to H in the C-domain. An extra helix at the N terminus is named N. The sites in the N-domain are Ca 2ϩ -specific and important for the regulation of muscle contract...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Calcium-dependent Changes in the Flexibility of the Regulatory Domain of Troponin C in the Troponin Complex

Blumenschein

Stone

Fletterick³

et al. 2005

Journal of Biological Chemistry

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“…Although the relative dispositions of individual subdomains in the crystal structure, including the somewhat compact cTnC, were found to be consistent with the earlier SANS-derived model, 51 this crystal structure provided the opportunity for the first high-resolution structural interpretation of the Ca 21 -regulatory mechanism. Using this cTn crystal structure as the starting frame and a rigid body Monte-Carlo procedure to refine against a full set of SANS and SAXS data from sTn, King et al 61 generated atomic-resolution models to represent the sTnC-sTnI-sTnT2 ternary complex in Ca 21 -free and Ca 21 -bound states. Experimentally, the researchers collected SANS data from sTn complexes, in which all possible combinations of the subunits were deuterated, in both the Ca 21 -free and Ca 21 -bound states.…”

Section: Probing the Structures Of Muscle Regulatory Proteins 509mentioning

confidence: 99%

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

Jeffries

Trewhella

2011

Biopolymers

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Small‐angle X‐ray and neutron scattering with contrast variation have made important contributions in advancing our understanding of muscle regulatory protein structures in the context of the dynamic molecular processes governing muscle action. The contributions of the scattering investigations have depended upon the results of key crystallographic, NMR, and electron microscopy experiments that have provided detailed structural information that has aided in the interpretation of the scattering data. This review will cover the advances made using small‐angle scattering techniques, in combination with the results from these complementary techniques, in probing the structures of troponin and myosin binding protein C. A focus of the troponin work has been to understand the isoform differences between the skeletal and cardiac isoforms of this major calcium receptor in muscle. In the case of myosin binding protein C, significant data are accumulating, indicating that this protein may act to modulate the primary calcium signals from troponin, and interest in its biological role has grown because of linkages between gene mutations in the cardiac isoform and serious heart disease. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 505–516, 2011.

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“…The impact is also evident in the scientific press. In SANS, two recent examples in which labelling of selected sub-units has allowed quaternary structure to be determined have been published by King et al [85], and by Callow et al [23]. In the former case, selective deuteration of a full set of ternary complexes of troponin has been used to study the large structural change that occurs upon calcium binding and to identify the sub-unit in which this change principally takes place.…”

Section: Deuteration Laboratoriesmentioning

confidence: 99%

New sources and instrumentation for neutrons in biology

et al. 2008

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Solution Structure of the Chicken Skeletal Muscle Troponin Complex Via Small-angle Neutron and X-ray Scattering

Cited by 39 publications

References 72 publications

Calcium-dependent Changes in the Flexibility of the Regulatory Domain of Troponin C in the Troponin Complex

Calcium-dependent Changes in the Flexibility of the Regulatory Domain of Troponin C in the Troponin Complex

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

New sources and instrumentation for neutrons in biology

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