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Ca2+-Induced Conformational Change of Troponin C from the Japanese Pearl Oyster, Pinctada fucata

scientific research. 2018;

Daisuke Funabara*, Daisuke Ishikawa, Yoshinori Urakawa, Satoshi Kanoh

Products/Services Used	Details	Operation
Gene Synthesis	We designed Pifuc-TnC-E142Q, a mutant variant lacking the ability to bind Ca2+ due to the substitution of a glutamic acid (E142) residue located in the site IV EF-hand motif to glutamine (Q) (Figure 1). DNA fragments encoding Pifuc-TnC or Pifuc-TnC-E142Q, with codon usage optimized for expression in Escherichia coli, were commercially synthesized by GenScript Gene Synthesis Service (GenScript, Piscataway, NJ, USA) and inserted into the T7 expression vector pET15b (Novagen Darmstadt, Germany), creating an in-frame N-terminal fusion of six histidine residues. E. coli BL21(DE3) transformed with pET-Pifuc-TnC or pET-Pifuc-TnC-E142Q were cultured in auto-induction media at 37˚C for 24 hours [12] . The cultured E. coli collected by centrifugation was suspended in a lysis buffer included in a kit EzBactYeast Crusher (ATTO, Tokyo, Japan). The supernatant of the lysate obtained by centrifugation containing Pifuc-TnC or Pifuc-TnC-E142Q was subjected to affinity chromatography with a Bio-Scale Mini Profinity IMAC cartridge (Bio-Rad, Hercules, CA, USA) under native conditions according to the manufacturer’s instructions. The purity of the eluted proteins was confirmed using SDS-PAGE and Coomassie blue staining. Protein concentrations were measured by the Bradford method using bovine serum albumin as a standard. Purified protein samples were freeze-dried following dialysis against 10 mM ammonium bicarbonate (pH 8.0).	Get A Quote

摘要

Troponin is a thin filament-associated regulator of vertebrate striated muscle contraction. Troponin changes its structure upon Ca2+ binding to troponin C, one of the subunits of troponin, allowing myosin to interact with actin. We recently elucidated the molecular characteristics of the Japanese pearl oyster Pinctada fucata troponin C (Pifuc-TnC), revealing the possibilities that Pifuc-TnC and vertebrate muscle TnC play dissimilar roles in muscle contraction. Pifuc-TnC has four EF-hand motifs, but, unlike vertebrate TnC, only one (site IV) was predicted to bind Ca2+. To confirm the number of Ca2+-binding sites in Pifuc-TnC and whether Ca2+ binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca2+-binding site of site IV), following their expression in laboratory E. coli. Isothermal titration calorimetry demonstrated Ca2+ binding to Pifuc-TnC, whereas Pifuc-TnC-E142Q was unable to bind Ca2+, confirming that site IV is the only Ca2+-binding site in Pifuc-TnC. Pifuc-TnC eluted in a later fraction from a gel filtration column in the presence of Ca2+ compared with the condition when Ca2+ was absent. In contrast, the elution profiles of Pifuc-TnC-E142Q were equivalent in both the presence and absence of Ca2+, suggesting that Ca2+ binding to Pifuc-TnC induces a conformational change that delays its elution from the column. UV-absorption spectral analysis revealed that binding of Ca2+ to Pifuc-TnC caused an increase in absorption at a wavelength of approximately 250 nm, possibly because phenylalanine residues had been exposed on the surface of the molecule as a result of a conformational change. Differential scanning calorimetric analyses of Pifuc-TnC showed aggregation in the presence of Ca2+ in accordance with an increase of temperature, but no aggregation was seen in the absence of Ca2+. In combination, these findings suggest that Ca2+ binding to site IV induces a conformational change in Pifuc-TnC.