Calcium-induced troponin flexibility revealed by distance distribution measurements between engineered sites.
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 TnI 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.
Zhao
X
X
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201, USA.
Kobayashi
T
T
Malak
H
H
Gryczynski
I
I
Lakowicz
J
J
Wade
R
R
Collins
J H
JH
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D011487:Protein Conformation; D014336:Troponin / Q000737:chemistry*
D014336:Troponin; D004533:Egtazic Acid; D000251:Adenosine Triphosphatases; D002118:Calcium