Critical minimum length of the central helix in troponin C for the Ca2+ switch in muscular contraction.

Babu, A.; Rao, Venu G.; Hong, Seonki; Gulati, Jagdish

doi:10.1016/s0021-9258(19)36504-4

Cited by 25 publications

(14 citation statements)

References 24 publications

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“…Figure 2 also shows that all mutants became more helical upon binding of Mg2+ to the high-affinity sites, with a small further increase upon binding of Ca2+ to the low-affinity sites. The a-helix, calculated from the ellipticity measured at 222 nm, increased to 44%-49% and 55%-58% upon binding to Mg2+ and Ca2+, respectively, comparable to published values (Kawasaki & van Eerd, 1972;Dobrowolski et al, 1991b;Babu et al, 1993). The slightly lower ellipicity of TnCinah compared to wild type TnC is most likely related to the difficulty to obtain highly accurate measurements of protein concentration.…”

Section: Resultssupporting

confidence: 84%

“…In addition, we made a deletion mutant lacking the entire D/E linker (three turns). Previous work showed that deletion of up to two turns (seven residues) did not greatly affect the regulatory function (Dobrowolski et al, 1991 a,b;Sheng et al, 1991;Babu et al, 1993). However, Babu et al (1993) showed deletion of more than seven residues results in serious loss of function as tested on skinned muscle fibers.…”

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confidence: 96%

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Investigation of the Structural Requirements of the Troponin C Central Helix for Function

Ramakrishnan¹,

Hitchcock‐DeGregori²

1995

Biochemistry

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The two globular Ca(2+)-binding domains of troponin C are connected by a three-turn, exposed central helix. The requirements of this helical linker for regulatory function are not fully understood. In the present work we investigated the structural requirement of the linker using a series of insertion mutations that differ in predicted flexibility. TnCinrc has a nine-residue flexible random coil insert, TnCinpp has a nine-residue rigid polyproline insert (three turns), and TnCin alpha h has a seven-residue insert with high potential of forming alpha-helix. TnCinrc and TnCinpp were defective in the activation of the regulated actomyosin ATPase activity in the presence of Ca2+ when compared to wild type or TnCin alpha h, suggesting that altering the flexibility of the central helix impairs the regulatory function of troponin C. TnCin alpha h, TnCinrc, and TnCinpp had 87% +/- 3, 62% +/- 3, and 58% +/- 2 of the wild type activity, respectively (n = 6). All insertions in the central helix resulted in elongation of molecule compared to wild type TnC as determined by Stokes' radius. The Ca(2+)-affinity, the Ca(2+)-dependence of the actomyosin ATPase, and the stability of the insertion mutants were similar to wild type. Deletions of up to two turns of the central helix have little effect on troponin C function [Dobrowolski, Z., Xu, G-Q., & Hitchcock-DeGregori, S. E. (1991) J. Biol. Chem. 266, 5703-5710]. In another mutant (TnCd11) the entire central helix, 87KEDAKGKSEEE97, was deleted. With TnCd11, activation of the actomyosin ATPase activity in the presence of Ca2+ was normal, but inhibition in the absence of Ca2+ was less effective. Interaction of TnCd11 with TnI was altered. There was a 2-fold excess of TnCd11 in reconstituted Tn complex, consistent with another report [Babu, A., Rao, V. G, Su, H., & Gulati, J. (1993) J. Biol. Chem. 268, 19232-19238]. Our results suggest that the native length and structure of the central helix are optimal for normal regulatory function and that connectivity alone is insufficient for TnC function.

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Section: Resultssupporting

confidence: 84%

mentioning

confidence: 96%

Investigation of the Structural Requirements of the Troponin C Central Helix for Function

Ramakrishnan¹,

Hitchcock‐DeGregori²

1995

Biochemistry

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“…Studies aimed toward elucidating the functionality of this linker utilized truncations of the skTnC linker, which impaired its flexibility and regulation of the actomyosin ATPase. 32–34 In calmodulin (CaM), transient interdomain association leads to compact states and is similarly modulated by the length and rigidity of the central linker. 35 In the context of TnC's binding partner TnI, when cTnI 1–80 is bound to cardiac C-TnC, the D/E linker binds the inhibitory cTnI peptide, iTnI 129–149 .…”

Section: Discussionmentioning

confidence: 99%

Anomalous structural dynamics of minimally frustrated residues in cardiac troponin C triggers hypertrophic cardiomyopathy

et al. 2021

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“…(1) Mutagenesis. The mutants were generated from the cDNA encoding rabbit fast-twitch skeletal muscle TnC, as described previously (Babu et al, 1993;Babu et al, 1992). The gene was cloned in the pT7 plasmid and included 22 unique restriction sequences which greatly facilitated the mutagenesis.…”

Section: Methods (A) Experimental Proceduresmentioning

confidence: 99%

“…In contrast to CaM, the function of TnC as an on/off switch for muscular contraction seems to depend on the precise separation between the two lobes during the complexation of TnC with Tnl (Babu et al, 1993;Keleti et al, 1994;Olah & Trewhella, 1994). Therefore, the present comparative analysis of functional and molecular properties of WT sTnC and its variants centers on the properties that are likely to determine the differences between TnC and CaM in their mechanisms of interaction with targets.…”

mentioning

confidence: 97%

Functional Role of Arginine-11 in the N-Terminal Helix of Skeletal Troponin C: Combined Mutagenesis and Molecular Dynamics Investigation

Gulati¹,

Akella²,

Hong³

et al. 1995

Biochemistry

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The two main structural differences between calmodulin (CaM) and skeletal troponin C (sTnC) are the absence in CaM of (i) the short N-terminal helix in TnC and (ii) the triplet KGK (residues 91-93; numbering according to chicken sTnC). It was recently shown that deletion of both structural groups from sTnC imparted to the resulting construct the CaM-like ability to activate phosphodiesterase (PDE) and to regulate force development in smooth muscle. To continue probing of the structural basis of the differential behavior of sTnC and CaM, residue Arg-11 in rabbit sTnC was mutated to Ala because the interactions of Arg-11 with distal residues in the N-terminal domain seem to link the N-terminal helix to the rest of the structure. The mutant exhibits CaM-like function in its ability to activate PDE (about 50% of CaM at 5 microM concentration). If, in addition, the KGK triplet is also deleted, PDE activation increases to about 80%. Both constructs retain their TnC function to nearly 100%. To explore the mechanistic basis of this remarkable observation, computational simulations of the molecular dynamics (MD) were carried out for both wild-type 4Ca2+.sTnC and the 4Ca2+.R11A mutant, and the results were compared to those from earlier simulations of 4Ca2+.CaM. Two types of structural changes observed from such simulations of the molecular dynamics of CaM had been considered to have a functional role: (i) a compaction to a more globular form and (ii) a reorientation of the Ca-binding domains around the central tether helix.(ABSTRACT TRUNCATED AT 250 WORDS)

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Critical minimum length of the central helix in troponin C for the Ca2+ switch in muscular contraction.

Cited by 25 publications

References 24 publications

Investigation of the Structural Requirements of the Troponin C Central Helix for Function

Investigation of the Structural Requirements of the Troponin C Central Helix for Function

Anomalous structural dynamics of minimally frustrated residues in cardiac troponin C triggers hypertrophic cardiomyopathy

Functional Role of Arginine-11 in the N-Terminal Helix of Skeletal Troponin C: Combined Mutagenesis and Molecular Dynamics Investigation

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