Spectroscopic and ITC study of the conformational change upon Ca2+-binding in TnC C-lobe and TnI peptide complex from Akazara scallop striated muscle |
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Authors: | Yumoto Fumiaki Tanaka Hiroyuki Nagata Koji Miyauchi Yumiko Miyakawa Takuya Ojima Takao Tanokura Masaru |
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Institution: | a Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan b Department of Physiology II, Jikei University School of Medicine, Tokyo, Japan c Laboratory of Biochemistry and Biotechnology, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan |
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Abstract: | Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca2+ ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca2+-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC C-lobe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca2+-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca2+-binding properties of the complex of TnC C-lobe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca2+-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the C-lobe of scallop TnC. We therefore assumed that the effect of Ca2+-binding must lead to a change in the interaction mode between the C-lobe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca2+ signal to TnI in Tn ternary complex. |
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Keywords: | Tn troponin TnC troponin C TnI troponin I TnT troponin T TnC N-lobe TnC N-terminal lobe TnC C-lobe TnC C-terminal lobe ITC isothermal titration microcalorimetry NMR nuclear magnetic resonance CD circular dichroism TCA trichloroethanoic acid EDTA ethylenediaminetetraacetic acid MOPS 3-morpholinopropanesulfonic acid PIPES piperazine-1 4-bis(2-ethanesulfonic acid) |
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