Holtzer M E, Crimmins D L, Holtzer A
Department of Chemistry, Washington University, St. Louis, MO 63130.
Biopolymers. 1995 Jan;35(1):125-36. doi: 10.1002/bip.360350113.
The native tropomyosin molecule is a parallel, registered, alpha-helical coiled coil made from two 284-residue chains. Long excised subsequences (> or = 95 residues) form the same structure with comparable thermal stability. Here, we investigate local stability using shorter subsequences (20-50 residues) that are chemically synthesized or excised from various regions along the protein chain. Thermal unfolding studies of such shorter peptides by CD in the same solvent medium used in extant studies of the parent protein indicate very low helix content, almost no coiled-coil formation, and high thermal lability of such secondary structure as does form. This behavior is in stark contrast to extant data on leucine-zipper peptides and short "designed" synthetic peptides, many of which have high alpha-helix content and form highly stable coiled coils. The existence of short coiled coils calls into question the older idea that short subsequences of a protein have little structure. The present study supports the older view, at least in its application to tropomyosin. The intrinsic local alpha-helical propensity and helix-helix interaction in this prototypical alpha-helical protein is sufficiently weak as to require not only dimerization, but macro-molecular amplification in order to attain its native conformation in common benign media near neutral pH.
天然原肌球蛋白分子是由两条含284个残基的链构成的平行、对齐的α-螺旋卷曲螺旋。长的切除后序列（≥95个残基）形成相同的结构，且具有相当的热稳定性。在此，我们使用从蛋白质链的各个区域化学合成或切除的较短序列（20 - 50个残基）来研究局部稳定性。在用于亲本蛋白质现有研究的相同溶剂介质中，通过圆二色性对这些较短肽进行热变性研究表明，其螺旋含量非常低，几乎没有卷曲螺旋形成，并且所形成的这种二级结构具有高热不稳定性。这种行为与关于亮氨酸拉链肽和短的“设计”合成肽的现有数据形成鲜明对比，其中许多肽具有高α-螺旋含量并形成高度稳定的卷曲螺旋。短卷曲螺旋的存在对蛋白质短序列几乎没有结构这一旧观点提出了质疑。本研究支持旧观点，至少在其应用于原肌球蛋白方面是如此。在这种典型的α-螺旋蛋白中，固有的局部α-螺旋倾向和螺旋-螺旋相互作用足够弱，以至于不仅需要二聚化，还需要大分子扩增才能在接近中性pH的普通良性介质中达到其天然构象。
