Troponin organization on relaxed and activated thin filaments revealed by electron microscopy and three-dimensional reconstruction

The steric model of muscle regulation holds that at low Ca2+ concentration, tropomyosin strands, running along thin filaments, are constrained by trop onin in an inhibitory position that blocks myosin-binding sites on actin. C a2+ activation, releasing this constraint, allows tropomyosin movement, ini tiating actin-myosin interaction and contraction. Although the different po sitions of tropomyosin on the thin filament are well documented, correspond ing information on troponin has been lacking and it has therefore not been possible to test the model structurally. Here, we show that troponin can be detected on thin filaments and demonstrate how its changing association wi th actin can control tropomyosin position in response to Ca2+ To accomplish this, thin filaments were reconstituted with an engineered short tropomyos in, creating a favorable troponin stoichiomtery and symmetry for three-dime nsional analysis. We demonstrate that in the absence of Ca2+, troponin boun d to both tropomyosin and actin can act as a latch to constrain tropomyosin in a position on actin that inhibits actomyosin ATPase. In addition, we fi nd that on Ca2+ activation the actin-troponin connection is broken, allowin g tropomyosin to assume a second position, initiating actomyosin ATPase and thus permitting contraction to proceed. (C) 2001 Academic Press.