Production, crystallization, and preliminary X‐ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1‐47) of troponin I

Saijo, Yumiko; Takeda, Soichi; Scherer, Anna; Kobayashi, Tomoyoshi; Maéda, Yuichiro; Taniguchi, Hiroyuki; Yao, Ming; Wakatsuki, Soichi

doi:10.1002/pro.5560060420

Cited by 10 publications

(3 citation statements)

References 21 publications

(14 reference statements)

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“…More attention has been given to the biologically important interaction between TnC and the N‐terminal region of TnI [Ngai and Hodges, 1992; Sheng et al, 1992; Farah et al, 1994; Krudy et al, 1994; Dong et al, 1997; Saijo et al, 1997; Tripet et al, 1997; Van Eyk et al, 1997; Filatov et al, 1998; Vassylyev et al, 1998a,b; Abbott et al, 2000; Mercier et al, 2000] since it was first identified by Syska et al [1976]. In our previous investigation, we have shown that synthetic peptides corresponding to the N‐terminus of TnI were able to interact with TnC and prevent TnC from neutralizing TnI or TnI inhibitory peptide (Ip) induced inhibition of acto‐S1‐TM ATPase activity.…”

Section: Discussionmentioning

confidence: 99%

Structural and functional studies on Troponin I and Troponin C interactions

Ngai

Hodges

2001

J of Cellular Biochemistry

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Troponin I (TnI) peptides (TnI inhibitory peptide residues 104-115, Ip; TnI regulatory peptide resides 1-30, TnI1-30), recombinant Troponin C (TnC) and Troponin I mutants were used to study the structural and functional relationship between TnI and TnC. Our results reveal that an intact central D/E helix in TnC is required to maintain the ability of TnC to release the TnI inhibition of the acto-S1-TM ATPase activity. Ca(2+)-titration of the TnC-TnI1-30 complex was monitored by circular dichroism. The results show that binding of TnI1-30 to TnC caused a three-folded increase in Ca(2+) affinity in the high affinity sites (III and IV) of TnC. Gel electrophoresis and high performance liquid chromatography (HPLC) studies demonstrate that the sequences of the N- and C-terminal regions of TnI interact in an anti-parallel fashion with the corresponding N- and C-domain of TnC. Our results also indicate that the N- and C-terminal domains of TnI which flank the TnI inhibitory region (residues 104 to 115) play a vital role in modulating the Ca(2+)- sensitive release of the TnI inhibitory region by TnC within the muscle filament. A modified schematic diagram of the TnC/TnI interaction is proposed.

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Section: Discussionmentioning

confidence: 99%

Structural and functional studies on Troponin I and Troponin C interactions

Ngai

Hodges

2001

J of Cellular Biochemistry

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“…Before the availability of crystal structures of actin and myosin subfragment 1, for example, distances derived from RET measurements were used to deduce the spatial arrangements of certain residues in these proteins (e.g., Botts et al, 1989;Kekic et al, 1996;Miki et al, 1992, and references therein;Park et al, 1991). Although a complex of an Nterminal fragment of TnI and TnC has recently been crystallized (Saijo et al, 1997), no crystal of Tn or of a complex containing intact TnI has been available for x-ray diffraction. We (Tao et al, 1989) and others (Wang and Cheung, 1986) have used RET to measure distances between various sites in these complexes.…”

Section: Introductionmentioning

confidence: 99%

Localization of Cys133 of Rabbit Skeletal Troponin-I with Respect to Troponin-C by Resonance Energy Transfer

Luo

Gergely

et al. 1998

Biophysical Journal

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We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys133 of troponin-I (TnI) with respect to troponin-C (TnC) in the ternary troponin complex and the binary TnC.TnI complex in the presence and absence of Ca2+. Cys133 of TnI was chosen because our previous work has shown that the region of TnI containing this residue undergoes Ca2+-dependent movements between actin and TnC, and may play an important role in the regulatory function of troponin. For this purpose, a TnI mutant with a single Cys at position 133, and TnC mutants, each with a single Cys at positions 5, 12, 21, 41, 49, 89, 98, 133, and 158, were constructed by site-directed mutagenesis. The distances between TnI Cys133 and each of the nine residues in TnC were then measured. Using a least-squares minimization procedure, we determined the position of TnI Cys133 in the coordinate system of the crystal structure of TnC. Our results show that in the presence of Ca2+, TnI Cys133 is located near residue 12 beneath the N-terminal lobe of TnC, and moves away by 12.6 A upon the removal of Ca2+. TnI Cys133 and the region of TnC that undergoes major change in conformation in response to Ca2+ are located roughly on opposite sides of TnC's central helix. This suggests that the region in TnI that undergoes Ca2+-dependent interaction with TnC is distinct from that interacting with actin.

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“…The alternative is expression in insect cells which can produce expression levels of 120-200 mg/L of infected cells using special enhancer sequences (16). E. coli cells are prokaryotic and thus generally do not acetylate the N-terminus of eukaryotic proteins although there are exceptions (17). Tm expressed in E. coli is not acetylated and will be largely inactive (14,18).…”

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Functional Homodimers and Heterodimers of Recombinant Smooth Muscle Tropomyosin

2006

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Skeletal and smooth muscle tropomyosin (Tm) require acetylation of their N termini to bind strongly to actin. Tm containing an N-terminal alanine serine (AS) extension to mimic acetylation, has been widely used to increase binding. The current study investigates the ability of an N-terminal AS extension to mimic native acetylation for both αα and ββ smooth Tm homodimers. We show that : 1) ASα-Tm binds actin 100 fold tighter than α-Tm, and 2 fold tighter than native smooth αβ-Tm; 2) β-Tm requires an AS extension to bind actin; 3) ASβ-Tm binds actin 10 fold weaker than ASα-Tm. Tm is present in smooth muscle tissues as >95% heterodimer, therefore we studied the binding of recombinant αβ heterodimers with different AS extensions. This study shows that recombinant Tm requires an AS extension on both α and β chains to bind like native Tm and that the α chain contributes more to actin binding than the β chain. Once assembled onto an actin filament all smooth muscle Tms regulate S1 binding to actin Tm in the same way, irrespective of the presence of an AS extension.Tropomyosin (Tm) is an actin binding, α-helical, coiled coil protein dimer which binds along the length of actin filaments in both muscle and non-muscle cells, and thus cooperatively regulates the interaction of actin with myosin heads. Muscle cells contain Tm expressed via two genes Tm1 and Tm2 (α and β), and isoform diversity (smooth and skeletal) results from the alternative splicing of the two genes. Each monomer of the dimer coiled-coil contains 284 amino acid residues (1-3).In skeletal muscle tissue Tm is present predominantly as a mixture of αβ heterodimer and αα homodimer (4-6). ββ is less stable and is much less common in muscle tissues (5,6). Studies involving guanidinium hydrochloride dissociation of native homodimer chains and anion exchange chromatography have shown that smooth muscle contains roughly equal amounts of α and β chains, more than 95% of which is present as heterodimer (7-9). Thermal unfolding studies also show that the heterodimer is preferentially formed at temperatures below its unfolding transition (9). Preference of the heterodimer is not fully understood, however viscosity and thermal unfolding measurements indicate that the strength of the end-to-end interaction of the smooth αβ heterodimer is much higher than that of the αα and ββ homodimers which have about the same strength of end-to-end interaction (7,8,10). The strength of end-toend interactions in the heterodimer might affect the cooperative behaviour of the tropomyosin, and thus might provide a functional advantage for the predominance of heterodimer in native smooth muscle. The ability to be able to assemble in vitro heterodimers would be very valuable. Such ability could be used to monitor the behaviour of a single α or β chain on the dimer via

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Production, crystallization, and preliminary X‐ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1‐47) of troponin I

Cited by 10 publications

References 21 publications

Structural and functional studies on Troponin I and Troponin C interactions

Structural and functional studies on Troponin I and Troponin C interactions

Localization of Cys133 of Rabbit Skeletal Troponin-I with Respect to Troponin-C by Resonance Energy Transfer

Functional Homodimers and Heterodimers of Recombinant Smooth Muscle Tropomyosin

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