Synthesis of a model protein of defined secondary and quaternary structure. Effect of chain length on the stabilization and formation of two-stranded alpha-helical coiled-coils.

Abstract

Five polyheptapeptides (Ac-(Lys-Leu-Glu-Ala-Leu-Glu-Gly)n-Lys-amide, where n = 1-5) of 8, 15, 22, 29, and 36 residues were synthesized by the solid-phase method. The peptides were purified by reversed-phase high-performance liquid chromatography. The ability of these peptides to form two-stranded alpha-helical coiled-coils in benign medium (1.1 M KC1, 0.05 M PO4 buffer, pH 7.0) was monitored by molecular weight determinations and circular dichroism studies and their physical properties were compared to carboxamidomethylated alpha-tropomyosin at cysteine 190 (CM-tropomyosin). The peptides TM-8, TM-15, and TM-22 were shown to be monomeric in both denaturant (8 M urea) and benign medium by gel-filtration high-performance liquid chromatography on TSK G2000 SW while peptides TM-29 and TM-36 were shown to be dimeric in benign medium both by gel-filtration and sedimentation equilibrium experiments. The CD spectra of the polyheptapeptides TM-8, TM-15, and TM-22 show large increases in molar ellipticity at 220 nm on the addition of trifluoroethanol (helix-inducing solvent) to the benign buffer. By comparison, the two-stranded polyheptapeptides (TM-29 and TM-36) and CM-tropomyosin do not show any increase in molar ellipticity at 220 nm. The helicity of polyheptapeptides increases with increasing chain length, with TM-36 having a value comparable with CM-tropomyosin [( theta]220 = -31,800 degrees and -32,200 degrees, respectively) which is considered to be essentially 100% alpha-helical. These small two-stranded alpha-helical coiled-coils are considerably more stable to temperature and urea denaturation than CM-tropomyosin. Whereas CM-tropomyosin is almost completely denatured in the presence of 6 M urea, TM-29 and TM-36 maintain 22 and 70% of their helicity, respectively. The 30% denaturation values (t30) are 74, 62, and 37 degrees C for TM-36, TM-29, and CM-tropomyosin, respectively, in benign medium (1.1 M KC1:PO4 buffer, pH 7.0). The t30 values can be substantially decreased in the presence of denaturant (3 M urea, 0.1 M KC1, PO4 buffer, pH 7.0) to 62 and 43 degrees C for TM-36 and TM-29, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)