TY - JOUR
T1 - Calcium-induced troponin flexibility revealed by distance distribution measurements between engineered sites
AU - Zhao, X.
AU - Kobayashi, T.
AU - Malak, H.
AU - Gryczynski, I.
AU - Lakowicz, J.
AU - Wade, R.
AU - Collins, J. H.
PY - 1995
Y1 - 1995
N2 - The contraction of vertebrate striated muscle is regulated by Ca2+ binding to troponin C (TnC). This causes conformational changes which alter the interaction of TnC with the inhibitory protein TnI and the tropomyosin- binding protein TnT. We have used the frequency domain method of fluorescence resonance energy transfer to measure TnT-TnC and TnT-TnI distances and distance distributions, in the presence of Ca2+, Mg2+, or EGTA, in TnC · TnI · TnT complexes. We reconstituted functional, ternary troponin complexes using the following recombinant subunits whose sequences were based on those of rabbit skeletal muscle: wild-type TnC; TnT25, a mutant C-terminal 25- kDa fragment of TnT containing a single Trp212 which was used as the sole donor for fluorescence energy transfer measurements; Trp-less ThI mutants which contained either no Cys or a single Cys at position 9, 96, or 117. Energy acceptor groups were introduced into TnC or TnI by labeling with dansyl aziridine or N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our results indicate that the troponin complex is relatively rigid in relaxed muscle, but becomes much more flexible when Ca2+ binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, releasing the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distribution measurements between troponin subunits.
AB - The contraction of vertebrate striated muscle is regulated by Ca2+ binding to troponin C (TnC). This causes conformational changes which alter the interaction of TnC with the inhibitory protein TnI and the tropomyosin- binding protein TnT. We have used the frequency domain method of fluorescence resonance energy transfer to measure TnT-TnC and TnT-TnI distances and distance distributions, in the presence of Ca2+, Mg2+, or EGTA, in TnC · TnI · TnT complexes. We reconstituted functional, ternary troponin complexes using the following recombinant subunits whose sequences were based on those of rabbit skeletal muscle: wild-type TnC; TnT25, a mutant C-terminal 25- kDa fragment of TnT containing a single Trp212 which was used as the sole donor for fluorescence energy transfer measurements; Trp-less ThI mutants which contained either no Cys or a single Cys at position 9, 96, or 117. Energy acceptor groups were introduced into TnC or TnI by labeling with dansyl aziridine or N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our results indicate that the troponin complex is relatively rigid in relaxed muscle, but becomes much more flexible when Ca2+ binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, releasing the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distribution measurements between troponin subunits.
UR - http://www.scopus.com/inward/record.url?scp=0029039847&partnerID=8YFLogxK
U2 - 10.1074/jbc.270.26.15507
DO - 10.1074/jbc.270.26.15507
M3 - Article
C2 - 7797544
AN - SCOPUS:0029039847
SN - 0021-9258
VL - 270
SP - 15507
EP - 15514
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -