Zhou N E, Kay C M, Hodges R S
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Mol Biol. 1994 Apr 8;237(4):500-12. doi: 10.1006/jmbi.1994.1250.
The role of interchain ionic interactions in controlling protein folding and stability has been studied by using de novo designed synthetic two-stranded alpha-helical coiled-coils. The model coiled-coil (denoted as EK) consists of two identical 35-residue polypeptide chains with a heptad repeat KgLaGbAcLdEeKf and a Cys residue at position 2 and an Ala residue at position 16 in each chain. The Lys residues at positions "g" in one chain and Glu residues at positions "e" in the other chain are expected to form interchain ion-pairs in the coiled-coil structure. This peptide forms a stable coiled-coil structure in benign medium (50 mM KCl, 25 mM PO4, pH7) with a [urea]1/2 value of 3.5 M. In contrast, two peptide analogs EE (EgLaGbAcLdEeKf) and KK (KgLaGbAcLdKeEf), which differ from EK in that EE contains only negatively charged Glu residues and KK contains only positively charged Lys residues at both positions e and g, each show a random coil structure in benign buffer. However, peptide EE or KK can form a stable coiled-coil structure if the interchain ionic repulsions are effectively suppressed either by changing pH or by using high salt concentrations. An equimolar mixture of these two peptides displays 100% alpha-helical content under the same conditions. These results demonstrate that although the interhelical ionic attractions are not essential for coiled-coil formation, a large number of these weak interactions can play an important role in the assembly of helices. Though interhelical ionic repulsions destabilize the homo-stranded coiled-coil, electrostatic attractions may stabilize the hetero-stranded coiled-coil. In addition, this study also suggests that the folding process for these synthetic model coiled-coils does not involve a single-stranded alpha-helix as a significantly populated folding intermediate.
通过使用从头设计的合成双链α-螺旋卷曲螺旋，研究了链间离子相互作用在控制蛋白质折叠和稳定性中的作用。模型卷曲螺旋（记为EK）由两条相同的35个残基的多肽链组成，具有七肽重复序列KgLaGbAcLdEeKf，每条链的第2位为半胱氨酸残基，第16位为丙氨酸残基。一条链中“g”位的赖氨酸残基和另一条链中“e”位的谷氨酸残基预计在卷曲螺旋结构中形成链间离子对。该肽在良性介质（50 mM KCl，25 mM PO4，pH7）中形成稳定的卷曲螺旋结构，[尿素]1/2值为3.5 M。相比之下，两种肽类似物EE（EgLaGbAcLdEeKf）和KK（KgLaGbAcLdKeEf）与EK的不同之处在于，EE在e和g位仅含有带负电荷的谷氨酸残基，KK在e和g位仅含有带正电荷的赖氨酸残基，它们在良性缓冲液中均呈现无规卷曲结构。然而，如果通过改变pH或使用高盐浓度有效地抑制链间离子排斥作用，肽EE或KK可以形成稳定的卷曲螺旋结构。在相同条件下，这两种肽的等摩尔混合物显示出100%的α-螺旋含量。这些结果表明，虽然螺旋间离子吸引力对于卷曲螺旋的形成不是必需的，但大量这些弱相互作用可以在螺旋组装中发挥重要作用。虽然螺旋间离子排斥会使同链卷曲螺旋不稳定，但静电吸引力可能会使异链卷曲螺旋稳定。此外，这项研究还表明，这些合成模型卷曲螺旋的折叠过程不涉及单链α-螺旋作为显著存在的折叠中间体。
