Lau S Y, Taneja A K, Hodges R S
J Biol Chem. 1984 Nov 10;259(21):13253-61.
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)
通过固相法合成了5种由8、15、22、29和36个残基组成的聚七肽（Ac-(Lys-Leu-Glu-Ala-Leu-Glu-Gly)n-Lys-酰胺，其中n = 1 - 5）。这些肽通过反相高效液相色谱进行纯化。通过分子量测定和圆二色性研究监测了这些肽在良性介质（1.1 M KCl，0.05 M磷酸缓冲液，pH 7.0）中形成双链α-螺旋卷曲螺旋的能力，并将它们的物理性质与半胱氨酸190处的羧酰胺甲基化α-原肌球蛋白（CM-原肌球蛋白）进行了比较。通过在TSK G2000 SW上进行凝胶过滤高效液相色谱，发现肽TM-8、TM-15和TM-22在变性剂（8 M尿素）和良性介质中均为单体，而肽TM-29和TM-36通过凝胶过滤和沉降平衡实验表明在良性介质中为二聚体。聚七肽TM-8、TM-15和TM-22的圆二色光谱显示，在良性缓冲液中加入三氟乙醇（螺旋诱导溶剂）后，220 nm处的摩尔椭圆率大幅增加。相比之下，双链聚七肽（TM-29和TM-36）和CM-原肌球蛋白在220 nm处的摩尔椭圆率没有任何增加。聚七肽螺旋度随链长增加而增加，TM-36的值与CM-原肌球蛋白相当（[θ]220分别为-31,800°和-32,200°），被认为基本上是100%α-螺旋。这些小的双链α-螺旋卷曲螺旋对温度和尿素变性的稳定性比CM-原肌球蛋白高得多。CM-原肌球蛋白在6 M尿素存在下几乎完全变性，而TM-29和TM-36分别保持其螺旋度的22%和70%。在良性介质（1.1 M KCl:磷酸缓冲液, pH 7.0）中，TM-36、TM-29和CM-原肌球蛋白的30%变性温度（t30）分别为74、62和37℃。在变性剂（3 M尿素，0.1 M KCl，磷酸缓冲液，pH 7.0）存在下，TM-36和TM-29的t30值可分别大幅降至62和43℃。（摘要截于400字）
