CONTROLLED FORMATION OF MODEL HOMODIMER AND HETERODIMER COILED-COIL POLYPEPTIDES
CONTROLLED FORMATION OF MODEL HOMODIMER AND HETERODIMER COILED-COIL POLYPEPTIDES
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DOI：
10.1021/bi00210a015
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发表时间：
1993-11-30
期刊：
影响因子：
2.9
通讯作者：
CHAIKEN, IM
中科院分区：
文献类型：
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作者：
GRADDIS, TJ;MYSZKA, DG;CHAIKEN, IM
合成了序列简化的卷曲螺旋多肽并表征了它们的折叠特性，以定义卷曲螺旋界面处带电边界残基对于同二聚体和异二聚体结构的受控形成的作用。设计三种肽以形成平行卷曲螺旋，其中缬氨酸和亮氨酸分别占据七肽重复序列abcdefg的疏水界面位置a和d。 命名为E/K42的多肽，具有七肽重复序列VSSLESK，在界面边界位置e和g中分别含有谷氨酸和赖氨酸，并被设计成在中性pH下形成卷曲螺旋同二聚体。另外两种多肽，命名为 E/E35 和 K/K35，分别具有七肽重复序列 VSSLESE 和 VSSLKSK。 E/E35 在 e 和 g 位均仅含有谷氨酸； K/K35，仅赖氨酸。 E/E35 和 K/K35 被设计为在中性 pH 下结合时形成稳定的卷曲螺旋异二聚体。所有三种多肽均通过固相合成制备，并通过反相高效液相色谱和尺寸排阻色谱纯化。通过圆二色性和尺寸排阻色谱法测定，E/K42 形成稳定的二聚体卷曲螺旋结构。 E/K42 的 α 螺旋含量在中性 pH 值时最高，在极端 pH 值时降低。微摩尔浓度下 E/K42 的 α 螺旋结构的 T(m) 为 62-65 摄氏度，并表现出与二聚体形成一致的热变性浓度依赖性。与 E/K42 的结果相反，E/E35 和 K/K35 的混合物（但两者都不单独）在中性 pH 下形成 α 螺旋。在微摩尔浓度下，E/E35:K/K35 混合物的 T(m) 为 60-63 摄氏度，并在凝胶过滤色谱中以二聚体形式洗脱，表明肽形成稳定的卷曲螺旋异二聚体。因此，对于两个肽，每个在所有 e 和 g 位置都具有单一类型的带电残基，但彼此带相反电荷，异二聚体可以分别通过电荷吸引和排斥来稳定，同二聚体则不稳定。为了支持这一结论，酸性多肽E/E35在低pH下形成α螺旋结构，而碱性多肽K/K35在高pH下形成α螺旋结构。结果表明，卷曲螺旋肽七肽重复的位置 e 和 g 可以改变以控制异二聚体和同二聚体的形成。
Sequence-simplified coiled coil polypeptides were synthesized and their folding properties characterized in order to define the role of charged border residues at the coiled coil interface for the controlled formation of homodimer and heterodimer structures. Three peptides were designed to form parallel coiled coils with valine and leucine occupying the hydrophobic interface positions a and d, respectively, of the heptad repeat abcdefg. The polypeptide designated E/K42, with the heptad repeat sequence VSSLESK, contained glutamate and lysine in the interface border positions e and g, respectively, and was designed to form a coiled coil homodimer at neutral pH. Two other polypeptides, designated E/E35 and K/K35, have the heptad repeats VSSLESE and VSSLKSK, respectively. E/E35 contains only glutamic acid at both e and g positions; K/K35, only lysine. E/E35 and K/K35 were designed to form a stable coiled coil heterodimer when combined at neutral pH. All three polypeptides were prepared by solid-phase synthesis and purified by reverse-phase high-performance liquid chromatography followed by size-exclusion chromatography. E/K42 formed a stable dimeric coiled coil structure as determined by circular dichroism and size-exclusion chromatography. The alpha-helical content of E/K42 was highest at neutral pH and decreased at extremes of pH. The alpha-helical structure of E/K42 at micromolar concentrations had a T(m) of 62-65-degrees-C and exhibited a concentration dependence of thermal denaturation consistent with dimer formation. In contrast to results with E/K42, a mixture of E/E35 and K/K35, but neither alone, forms alpha-helix at neutral pH. At micromolar concentrations the E/E35:K/K35 mixture had a T(m) of 60-63-degrees-C and eluted as a dimer in gel filtration chromatography, suggesting that the peptides form a stable coiled coil heterodimer. Hence, for two peptides, each with a single type of charged residue at all e and g positions but oppositely charged with respect to each other, heterodimers can be stabilized and homodimers destabilized by charge attraction and repulsion, respectively. In support of this conclusion, the acidic polypeptide E/E35 forms alpha-helical structure at low pH, while the basic polypeptide K/K35 forms alpha-helical structure at high pH. The results argue that positions e and g of the heptad repeat of coiled coil peptides can be varied to control heterodimer and homodimer formation.