|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1994 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
A cofilin active site analog dodecapeptide was obtained. Binding of the peptide to actin was characterized by two-dimensional proton NMR spectroscopy. The proton resonance of the peptide were unequivocally assigned. Although the Lys residues that are crosslinked to actin are located at the C-terminal portion of the peptide, intraresidue transferred NOEs were observed for all 12 amino acid residues of the peptide, indicating that the peptide interacts with actin along its entire length. The absence of interresidue TRNOEs for the C-terminal residues indicated that this part does not fold tightly, and that the overall folding is not essential for the activity of the peptide. In contrast, the N-terminal 5 residues exhibited strong interresidue TRNOEs. The N-terminal portion of the peptide is proposed to bind to actin by fprming a locally folded structure.
Mass purification procedures for cofilin and destrin, which are necessary for the determination of the overall tertiary structure, were developed.Each expression plasmid was transfected into E.Coli. The soluble fractions were obtained from the expression-induced cells. The proteins were purified by sequential chromatographies. Using the procedure, enough amount of destuin was obtained and used for NMR measurement. The complete tertiary structure of destrin, however, remained to be solved.
Domain structures of destrin and cofilin were studied by limited proteolysis, circular dichroism spectroscopy and secondary structure prediction. The two proteins were digested by three different proteases. Among them, chymotrypsin most effectively digested destrin, giving a stable fragment with an apparent molecular mass of 8kDa. A sequence analysis indicated that the fragment contains the actin binding sequence. Destrin was supposed to consist of two distinctive domains. The domain structure of cofilin was apparently different from that of destrin.