The Rates of Switching Movement of Troponin T between Three States of Skeletal Muscle Thin Filaments Determined by Fluorescence Resonance Energy Transfer

Shitaka, Y.; Kimura, Chieko; Miki, Masao

doi:10.1074/jbc.m408553200

Cited by 12 publications

(14 citation statements)

References 35 publications

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“…An influence of the strong cross-bridge on TnT position on the reconstituted thin filament was observed recently, 52 in agreement with our observations. A caveat with our observations is that TnT interactions with actin-Tm may be different when in near quaternary (actin-Tm-TnT) or quaternary (actinTm-TnCIT) form.…”

Section: Discussionsupporting

confidence: 93%

Myofibrillar Troponin Exists in Three States and there Is Signal Transduction along Skeletal Myofibrillar Thin Filaments

Swartz

Yang

Sen

et al. 2006

Journal of Molecular Biology

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Activation of striated muscle contraction is a highly cooperative signal transduction process converting calcium binding by troponin C (TnC) into interactions between thin and thick filaments. Once calcium is bound, transduction involves changes in protein interactions along the thin filament. The process is thought to involve three different states of actin-tropomyosin (Tm) resulting from changes in troponin's (Tn) interaction with actin-Tm: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interactions and favored by calcium binding to Tn, and an open or M state allowing strong myosin interactions. This was tested by measuring the apparent rate of Tn dissociation from rigor skeletal myofibrils using labeled Tn exchange. The location and rate of exchange of Tn or its subunits were measured by high-resolution fluorescence microscopy and image analysis. Three different rates of Tn exchange were observed that were dependent on calcium concentration and strong cross-bridge binding that strongly support the three-state model. The rate of Tn dissociation in the non-overlap region was 200-fold faster at pCa 4 (C-state region) than at pCa 9 (B-state region). When Tn contained engineered TnC mutants with weakened regulatory TnI interactions, the apparent exchange rate at pCa 4 in the non-overlap region increased proportionately with TnI-TnC regulatory affinity. This suggests that the mechanism of calcium enhancement of the rate of Tn dissociation is by favoring a TnI-TnC interaction over a TnI-actin-Tm interaction. At pCa 9, the rate of Tn dissociation in the overlap region (M-state region) was 100-fold faster than the non-overlap region (B-state region) suggesting that strong cross-bridges increase the rate of Tn dissociation. At pCa 4, the rate of Tn dissociation was twofold faster in the non-overlap region (C-state region) than the overlap region (M-state region) that likely involved a strong cross-bridge influence on TnT's interaction with actin-Tm. At sub-maximal calcium (pCa 6.2-5.8), there was a long-range influence of the strong cross-bridge on Tn to enhance its dissociation rate, tens of nanometers from the strong cross-bridge. These observations suggest that the three different states of actin-Tm are associated with three different states of Tn. They also support a model in which strong cross-bridges shift the regulatory equilibrium from a TnI-actin-Tm interaction to a TnC-TnI interaction that likely enhances calcium binding by TnC.

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Section: Discussionsupporting

confidence: 93%

Myofibrillar Troponin Exists in Three States and there Is Signal Transduction along Skeletal Myofibrillar Thin Filaments

Swartz

Yang

Sen

et al. 2006

Journal of Molecular Biology

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“…Fluorescent probes on actin (5), tropomyosin (16)(17)(18)(19), and troponin (7)(8)(9)20,21) report changes in state transitions. In this work, we focused on probes on tropomyosin because the position of tropomyosin on actin ultimately defines the activity of thin filaments.…”

Section: Introductionmentioning

confidence: 99%

Kinetics of Regulated Actin Transitions Measured by Probes on Tropomyosin

Borrego-Diaz

Chalovich

2010

Biophysical Journal

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Changes in the muscle regulatory protein complex, troponin, are important for modulation of activity and may occur as a result of disease-causing mutations. Both increases and decreases in the rate of ATP hydrolysis by myosin may occur as dictated by changes in the distribution of actin-tropomyosin-troponin among its different states. It is important to measure the rates of transition among these states to study physiological adaptation and disease processes. We show here that acrylodan or pyrene probes on tropomyosin can be used to monitor the transition from active to intermediate and inactive states of actin-tropomyosin-troponin. Transitions measured in the absence of calcium had two phases, as previously reported for some other probes on troponin and actin. The first step was a rapid equilibrium that favored the formation of the intermediate state and had an apparent rate constant less than that of S1-ATP dissociation. The second fluorescence transition was slower, with an apparent constant that increased from approximately 5 to 80/s over a range of 1-37 degrees C. Only the initial rapid transition was seen in the presence of saturating calcium. The acrylodan probe had the advantage of yielding a larger signal than the pyrene probe. Furthermore, the acrylodan signal decreased in going from the active state to the intermediate state, and then increased upon going to the inactive state.

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“…To date this has not been accomplished, nor to the best of our knowledge even attempted. However, numerous studies have attempted to simulate the physiological system’s Ca 2+ kinetics by studying TnC in isolation, in the TnC–TnI complex, in Tn and in regulated thin filaments (Johnson et al, 1981, 1994; Luo et al, 2002; Davis et al, 2004; Shitaka et al, 2004, 2005). We have taken these studies a step further and closer to the physiological system by utilizing isolated rigor skeletal myofibrils.…”

Section: Introductionmentioning

confidence: 99%

Measurement of Calcium Dissociation Rates from Troponin C in Rigor Skeletal Myofibrils

Little¹,

Tikunova²,

Norman³

et al. 2011

Front. Physio.

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Ca2+ dissociation from the regulatory domain of troponin C may influence the rate of striated muscle relaxation. However, Ca2+ dissociation from troponin C has not been measured within the geometric and stoichiometric constraints of the muscle fiber. Here we report the rates of Ca2+ dissociation from the N-terminal regulatory and C-terminal structural domains of fluorescent troponin C constructs reconstituted into rabbit rigor psoas myofibrils using stopped-flow technology. Chicken skeletal troponin C fluorescently labeled at Cys 101, troponin CIAEDANS, reported Ca2+ dissociation exclusively from the structural domain of troponin C at ∼0.37, 0.06, and 0.07/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Ca2+ dissociation from the regulatory domain was observed utilizing fluorescently labeled troponin C containing the T54C and C101S mutations. Troponin CMIANST54C,C101S reported Ca2+ dissociation exclusively from the regulatory domain of troponin C at >1000, 8.8, and 15/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Interestingly, troponin CIAANST54C,C101S reported a biphasic fluorescence change upon Ca2+ dissociation from the N- and C-terminal domains of troponin C with rates that were similar to those reported by troponin CMIANST54C,C101S and troponin CIAEDANS at all levels of the troponin C systems. Furthermore, the rate of Ca2+ dissociation from troponin C in the myofibrils was similar to the rate of Ca2+ dissociation measured from the troponin C-troponin I complexes. Since the rate of Ca2+ dissociation from the regulatory domain of TnC in myofibrils is similar to the rate of skeletal muscle relaxation, Ca2+ dissociation from troponin C may influence relaxation.

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The Rates of Switching Movement of Troponin T between Three States of Skeletal Muscle Thin Filaments Determined by Fluorescence Resonance Energy Transfer

Cited by 12 publications

References 35 publications

Myofibrillar Troponin Exists in Three States and there Is Signal Transduction along Skeletal Myofibrillar Thin Filaments

Myofibrillar Troponin Exists in Three States and there Is Signal Transduction along Skeletal Myofibrillar Thin Filaments

Kinetics of Regulated Actin Transitions Measured by Probes on Tropomyosin

Measurement of Calcium Dissociation Rates from Troponin C in Rigor Skeletal Myofibrils

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