CALCIUM-INDUCED TROPONIN FLEXIBILITY REVEALED BY DISTANCE DISTRIBUTION MEASUREMENTS BETWEEN ENGINEERED SITES

The contraction of vertebrate striated muscle is regulated by Ca2+ bin ding to troponin C (TnC). This causes conformational changes which alt er the interaction of TnC with the inhibitory protein TnI and the trop omyosin-binding protein TnT. We have used the frequency domain method of fluorescence resonance energy transfer to measure TnT-TnC and TnT-T nI 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 n- hose sequences were based on those of rabbit skeletal muscle: wild-ty pe TnC; TnT(25), a mutant C-terminal 25-kDa fragment of TnT containing a single Trp(212) which was used as the sole donor for fluorescence e nergy transfer measurements; Trp-less TnI: mutants which contained eit her 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 aziridi ne or N-(iodoacetyl)-N'-( 1-sulfo-5-naphthyl)ethylenediamine. Our resu lts indicate that the troponin complex is relatively rigid in relaxed muscle, but becomes much more flexible when Ca2+ binds to regulatory s ites 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 dist ance distribution measurements between troponin subunits.