Ca&sup&2+&/sup&-Induced Conformational Change of Troponin C from the Japanese Pearl Oyster, &i&Pinctada fucata&/i&

摘要

Troponin is a thin filament-associated regulator of vertebrate striated muscle contraction. Troponin changes its structure upon Ca ~( 2+ ) 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 Ca ~( 2+ ) . To confirm the number of Ca ~( 2+ ) -binding sites in Pifuc-TnC and whether Ca ~( 2+ ) binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca ~( 2+ ) -binding site of site IV), following their expression in laboratory E. coli . Isothermal titration calorimetry demonstrated Ca ~( 2+ ) binding to Pifuc-TnC, whereas Pifuc-TnC-E142Q was unable to bind Ca ~( 2+ ) , confirming that site IV is the only Ca ~( 2+ ) -binding site in Pifuc-TnC. Pifuc-TnC eluted in a later fraction from a gel filtration column in the presence of Ca ~( 2+ ) compared with the condition when Ca ~( 2+ ) was absent. In contrast, the elution profiles of Pifuc-TnC-E142Q were equivalent in both the presence and absence of Ca ~( 2+ ) , suggesting that Ca ~( 2+ ) binding to Pifuc-TnC induces a conformational change that delays its elution from the column. UV-absorption spectral analysis revealed that binding of Ca ~( 2+ ) 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 Ca ~( 2+ ) in accordance with an increase of temperature, but no aggregation was seen in the absence of Ca ~( 2+ ) . In combination, these findings suggest that Ca ~( 2+ ) binding to site IV induces a conformational change in Pifuc-TnC.