Engineering Competitive Magnesium Binding into the First EF-hand of Skeletal Troponin C

Davis, Jonathan P.; Rall, Jack A.; Reiser, Peter J.; Smillie, Lawrence B.; Tikunova, Svetlana B.

doi:10.1074/jbc.m208488200

Cited by 36 publications

(75 citation statements)

References 71 publications

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“…Similar Ca 2ϩ dissociation rates were measured in the absence of Mg 2ϩ (data not shown; summary in Table I). These results were consistent with the Ca 2ϩ dissociation rate from the regulatory domain of sTnC F29W not being affected by Mg 2ϩ (8), again indicative of competitive binding of Mg 2ϩ to the regulatory site of cTnC F27W .…”

Section: Measurements Of Ca 2ϩ Dissociation Rates Using Trp and Quin-supporting

confidence: 78%

“…To follow Ca 2 ϩ binding and exchange with the regulatory domain of cTnC, we utilized the intrinsic fluorescence of cTnC F27W , which has been previously used to study Ca 2ϩ binding to the N-domain site of cTnC and its mutants or isoforms (26 -29). This mutation is analogous to sTnC F29W , which enabled numerous studies of Ca 2ϩ and target protein binding to the N-domain of sTnC and its mutants (6,8,22,25,42).…”

Section: Using Acts To Estimate the Ca 2ϩ Association Rates To The N-mentioning

confidence: 99%

“…Mg 2ϩ -Because k off(Ca) is too fast to allow the experimental measurements of k on(Ca) using the standard pseudo-first order method in a stopped-flow apparatus (6), we estimated k on(Ca) by subjecting cTnC F27W and its mutants to ACTs of increasing duration and amplitude (8). The measurements were conducted in the presence of 3 or 12 mM Mg 2ϩ , to ensure complete Mg 2ϩ occupation of the C-domain sites.…”

Section: Using Acts To Estimate the Ca 2ϩ Association Rates To The N-mentioning

confidence: 99%

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Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C

Tikunova

Davis

2004

Journal of Biological Chemistry

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As anticipated, these selected hydrophobic residue substitutions increased the calcium affinity of the regulatory domain of cardiac troponin C F27W ϳ2.1-15.2-fold. Surprisingly, the increased calcium affinity caused by the hydrophobic residue substitutions was largely due to faster calcium association rates (2.6 -8.7-fold faster) rather than to slower calcium dissociation rates (1.2-2.9-fold slower). The regulatory N-domains of cardiac troponin C F27W and its mutants were also able to bind magnesium competitively and with physiologically relevant affinities (1.2-2.7 mM). The design of calcium-sensitizing cardiac troponin C mutants presented in this work enhances the understanding of how to control cation binding properties of EF-hand proteins and ultimately their structure and physiological function.

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Section: Measurements Of Ca 2ϩ Dissociation Rates Using Trp and Quin-supporting

confidence: 78%

Section: Using Acts To Estimate the Ca 2ϩ Association Rates To The N-mentioning

confidence: 99%

Section: Using Acts To Estimate the Ca 2ϩ Association Rates To The N-mentioning

confidence: 99%

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Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C

Tikunova

Davis

2004

Journal of Biological Chemistry

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“…Ca 2ϩ dissociation from the N-terminal regulatory domain of TnC F29W and its mutants when complexed with TnI 96 -148 were obtained utilizing Trp fluorescence excited at 275 nm with emission monitored through a UV transmitting black glass filter (UG1 from Oriel, Stratford, CT). k off was also measured using the fluorescent Ca 2ϩ chelator Quin-2 (6,8). Quin-2 was excited at 330 nm with its emission monitored through a 510-nm broad band pass interference filter (Oriel, Stratford, CT).…”

Section: Methodsmentioning

confidence: 99%

“…In conjunction with residues 96 -116, residues 117-148 of TnI are required for the complete inhibitory activity and regulatory interactions with actin and TnC (20 -23). Furthermore, the complete enhancement of the Ca 2ϩ sensitivity and the slowing of the Ca 2ϩ dissociation rate from the regulatory domain of TnC in the presence of intact TnI were mimicked by a peptide of TnI corresponding to residues 96 -148 (TnI 96 -148 ) (8). Thus, the Ca 2ϩ -dependent binding of the regulatory domain of TnC to TnI 96 -148 may be a good model system to study the Ca 2ϩ -dependent interactions between TnI and TnC that regulate muscle mechanics.…”

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confidence: 99%

Mutations of Hydrophobic Residues in the N-terminal Domain of Troponin C Affect Calcium Binding and Exchange with the Troponin C-Troponin I96–148 Complex and Muscle Force Production

Davis

Rall

Alionte

et al. 2004

Journal of Biological Chemistry

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Interactions between troponin C and troponin I play a critical role in the regulation of skeletal muscle contraction and relaxation. We individually substituted 27 hydrophobic Phe, Ile, Leu, Val, and Met residues in the regulatory domain of the fluorescent troponin C F29W with polar Gln to examine the effects of these mutations on: (a) the calcium binding and dynamics of troponin C F29W complexed with the regulatory fragment of troponin I (troponin I 96 -148 ) and (b) the calcium sensitivity of force production. Troponin I 96 -148 was an accurate mimic of intact troponin I for measuring the calcium dynamics of the troponin C F29W -troponin I complexes. The calcium affinities of the troponin C F29W -troponin I 96 -148 complexes varied ϳ243-fold, whereas the calcium association and dissociation rates varied ϳ38-and ϳ33-fold, respectively. Interestingly, the effect of the mutations on the calcium sensitivity of force development could be better predicted from the calcium affinities of the troponin C F29W -troponin I 96 -148 complexes than from that of the isolated troponin C F29W mutants. Most of the mutations did not dramatically affect the affinity of calcium-saturated troponin C F29W for troponin I 96 -148 . However, the Phe 26 to Gln and Ile 62 to Gln mutations led to >10-fold lower affinity of calcium-saturated troponin C F29W for troponin I 96 -148 , causing a drastic reduction in force recovery, even though these troponin C F29W mutants still bound to the thin filaments. In conclusion, elucidating the determinants of calcium binding and exchange with troponin C in the presence of troponin I provides a deeper understanding of how troponin C controls signal transduction.Troponin C (TnC) 1 regulates striated muscle contraction and relaxation through the binding and release of Ca 2ϩ (for review see Refs. 1-3). Skeletal muscle TnC (ϳ18 kDa) consists of globular N-and C-terminal domains connected by a 31-residue ␣-helix (for review see Refs. 4 and 5). Both domains bind two Ca 2ϩ ions through a pair of EF hand Ca 2ϩ -binding motifs. Each pair of EF hands interacts with one another through a short antiparallel ␤-sheet connecting the two Ca 2ϩ -binding loops (Ref. 6 and references within). The EF hands are numbered I-IV, and the helices flanking the loops are designated A-H, with an additional N-terminal 14-residue ␣-helix (Fig. 1, N helix), which is absent in the closely related EF hand Ca 2ϩ -binding protein calmodulin.Much is known about the cation binding properties of TnC in solution. Each EF hand system binds Ca 2ϩ and Mg 2ϩ competitively, with the two C-terminal EF hands possessing higher Ca 2ϩ and Mg 2ϩ affinities (6 -8). In fact, the Ca 2ϩ -binding sites of the C-domain of TnC possess ϳ10-fold higher Ca 2ϩ affinity with a greater than 100-fold slower Ca 2ϩ dissociation rate compared with those in the N-domain (6, 9). In part because of its high Ca 2ϩ and Mg 2ϩ affinities and slow Ca 2ϩ exchange rates (as compared with the kinetics of muscle contraction and relaxation), the C-domain is thought to play a struc...

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Clusters of bound Ca2+ initiate contraction in fast skeletal muscle

Brandt

Poggesi

2014

Archives of Biochemistry and Biophysics

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Engineering Competitive Magnesium Binding into the First EF-hand of Skeletal Troponin C

Cited by 36 publications

References 71 publications

Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C

Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C

Mutations of Hydrophobic Residues in the N-terminal Domain of Troponin C Affect Calcium Binding and Exchange with the Troponin C-Troponin I96–148 Complex and Muscle Force Production

Clusters of bound Ca2+ initiate contraction in fast skeletal muscle

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