The second half of the fourth period of tropomyosin is a key region for Ca(2+)-dependent regulation of striated muscle thin filaments.

Akiko Sakuma, Chieko Kimura-Sakiyama, Atsuhiro Onoue, Yuji Shitaka, Takahisa Kusakabe, Masao Miki

Department of Applied Chemistry and Biotechnology, Fukui University, 3-9-1 Bunkyo, Fukui 910-8507, Japan.

Biochemistry 2006 Aug 8

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Rabbit skeletal muscle alpha-tropomyosin (Tm), a 284-residue dimeric coiled-coil protein, spans seven actin monomers and contains seven quasiequivalent periods. X-ray analysis of cocrystals of Tm and troponin (Tn) placed the Tn core domain near residues 150-180 of Tm. To identify the Ca(2+)-sensitive Tn interaction site on Tm, we generated three Tm mutants to compare the consequences of sequence substitution inside and outside of the Tn core domain-binding region. Residues 152-165 and 156-162 in the second half of period 4 were replaced by corresponding residues 33-46 and 37-43 in the second half 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 were expressed in Escherichia coli. Both mTm152-165 and mTm156-162 suppressed the actin-activated myosin subfragment-1 Mg(2+)-ATPase rate regardless of whether Ca(2+) and Tn were present. On the other hand, mTm134-147 retained the normal Ca(2+)-sensitive regulation, although the actin binding of mTm alone was significantly impaired. Differential scanning calorimetry showed that the sequence substitution in the 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 for inducing conformational changes of the regulated thin filament required for its fully activated state.

Citation

Akiko Sakuma, Chieko Kimura-Sakiyama, Atsuhiro Onoue, Yuji Shitaka, Takahisa Kusakabe, Masao Miki. The second half of the fourth period of tropomyosin is a key region for Ca(2+)-dependent regulation of striated muscle thin filaments. Biochemistry. 2006 Aug 8;45(31):9550-8