| Abstract
| - Rabbit skeletal muscle α-tropomyosin (Tm), a 284-residue dimeric coiled-coil protein, spansseven actin monomers and contains seven quasiequivalent periods. X-ray analysis of cocrystals of Tmand troponin (Tn) placed the Tn core domain near residues 150−180 of Tm. To identify the Ca2+-sensitiveTn interaction site on Tm, we generated three Tm mutants to compare the consequences of sequencesubstitution inside and outside of the Tn core domain-binding region. Residues 152−165 and 156−162in the second half of period 4 were replaced by corresponding residues 33−46 and 37−43 in the secondhalf of period 1, respectively (termed mTm152−165 and mTm156−162, respectively), and residues 134−147 in the first half of period 4 were replaced with residues 15−28 in the first half of period 1 (mTm134−147). Recombinant Tms designed with an additional tripeptide, Ala-Ala-Ser, at the N-terminus wereexpressed in Escherichia coli. Both mTm152−165 and mTm156−162 suppressed the actin-activated myosinsubfragment-1 Mg2+-ATPase rate regardless of whether Ca2+ and Tn were present. On the other hand,mTm134−147 retained the normal Ca2+-sensitive regulation, although the actin binding of mTm alonewas significantly impaired. Differential scanning calorimetry showed that the sequence substitution inthe second half of period 4 affected the thermal stability of the complete Tm molecule and also the actin-induced stabilization. These results suggest that the second half of period 4 of Tm is a key region forinducing conformational changes of the regulated thin filament required for its fully activated state.
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