ABSTRACT
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.
Share and Cite:
Funabara, D. , Ishikawa, D. , Urakawa, Y. and Kanoh, S. (2018) Ca
2+-Induced Conformational Change of Troponin C from the Japanese Pearl Oyster,
Pinctada fucata.
American Journal of Molecular Biology,
8, 205-214. doi:
10.4236/ajmb.2018.84018.