The distance between Ca2+-binding site
III in the C-terminal domain and Cys35 in the N-terminal
domain in cardiac muscle troponin C (cTnC) was determined
with a single-tryptophan mutant using bound Tb3+
as the energy donor and iodoacetamidotetramethylrhodamine
linked to the cysteine residue as energy acceptor. The
luminescence of bound Tb3+ was generated through
sensitization by the tryptophan located in the 12-residue
binding loop of site III upon irradiation at 295 nm, and
this sensitized luminescence was the donor signal transferred
to the acceptor. In the absence of bound cation at site
II, the mean interdomain distance was found to be 48–49
Å regardless of whether the cTnC was unbound or bound
to cardiac troponin I, or reconstituted into cardiac troponin.
These results suggest that cTnC retains its overall length
in the presence of bound target proteins. The distribution
of the distances was wide (half-width >9 Å) and
suggests considerable interdomain flexibility in isolated
cTnC, but the distributions became narrower for cTnC in
the complexes with the other subunits. In the presence
of bound cation at the regulatory site II, the interdomain
distance was shortened by 6 Å for cTnC, but without
an effect on the half-width. The decrease in the mean distance
was much smaller or negligible when cTnC was complexed
with cTnI or cTnI and cTnT under the same conditions. Although
free cTnC has considerable interdomain flexibility, this
dynamics is slightly reduced in troponin. These results
indicate that the transition from the relaxed state to
an activated state in cardiac muscle is not accompanied
by a gross alteration of the cTnC conformation in cardiac
troponin.
