Zhou N E, Mant C T, Hodges R S
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Pept Res. 1990 Jan-Feb;3(1):8-20.
A nonpolar environment, such as the hydrophobic stationary phase of a reversed-phase chromatographic packing, may induce helical structures in potentially helical molecules. If a molecule becomes helical on binding and contains a preferred binding domain, as in the case of an amphipathic helix, then some residues may not be contributing to the same extent to the overall hydrophobicity of the peptide. Amphipathic alpha-helical structures may play an important role in protein folding and the interaction of amphipathic alpha-helices with a hydrophobic surface during RPC is likely to be a good mimic of their hydrophobic interactions with other hydrophobic regions in the folded protein. We have designed and synthesized two sets of model peptides of 7, 14, 21, 28 and 35 residues having the same composition but different sequences (Ac-Lys-Cys-Ala-Glu-Gly-Glu-Leu-[Lys-Leu-Glu-Ala-Gly-Glu-Leu]n-amide and Ac-Lys-Cys-Ala-Glu-Leu-Glu-Gly-[Lys-Leu-Glu-Ala-Leu-Glu-Gly]n-amide, where n = 1-4). Circular dichroism studies demonstrated that both sets of peptides had a high potential to form alpha-helical structure in a nonpolar environment, one set representing amphipathic alpha-helical structures and the other set representing non-amphipathic alpha-helical structures. Size-exclusion chromatography confirmed that all of the peptides in both sets were monomeric when bound to a reversed-phase matrix and also under the conditions used for circular dichroism measurements. Peptides with the same amino acid composition and similar secondary structure could be separated by reversed-phase chromatography. The difference in retention time between peptides of the same length increased with the peptide chain length, ranging from a difference of 2.9 min on a C8 column for the two 14-residue peptides up to a maximum difference of 7.3 min for the 35-residue peptides. From the observed and predicted retention times of these two sets of peptides during reversed-phase chromatography, we have demonstrated that it is possible not only to predict the retention behavior of amphipathic alpha-helices during reversed-phase chromatography, but also to deduce the presence of amphipathic alpha-helical structure in peptides based upon their retention data. If from studies such as these we are eventually able to predict, from only amino acid sequence information, the secondary structure of a peptide on binding to a hydrophobic matrix, we may be able to extrapolate this predictive facility to the conformation of the same sequence in larger polypeptides or proteins.
非极性环境，如反相色谱填料的疏水固定相，可能会在潜在的螺旋状分子中诱导出螺旋结构。如果一个分子在结合时形成螺旋结构且含有一个优先结合结构域，就像两亲性螺旋的情况那样，那么一些残基对肽的整体疏水性的贡献程度可能并不相同。两亲性α-螺旋结构可能在蛋白质折叠中起重要作用，并且在反相色谱过程中两亲性α-螺旋与疏水表面的相互作用可能很好地模拟了它们在折叠蛋白中与其他疏水区域的疏水相互作用。我们设计并合成了两组由7、14、21、28和35个残基组成的模型肽，它们具有相同的组成但序列不同（Ac-Lys-Cys-Ala-Glu-Gly-Glu-Leu-[Lys-Leu-Glu-Ala-Gly-Glu-Leu]n-酰胺和Ac-Lys-Cys-Ala-Glu-Leu-Glu-Gly-[Lys-Leu-Glu-Ala-Leu-Glu-Gly]n-酰胺，其中n = 1 - 4）。圆二色性研究表明，这两组肽在非极性环境中都具有形成α-螺旋结构的高潜力，一组代表两亲性α-螺旋结构，另一组代表非两亲性α-螺旋结构。尺寸排阻色谱证实，两组中的所有肽在与反相基质结合时以及在用于圆二色性测量的条件下都是单体。具有相同氨基酸组成和相似二级结构的肽可以通过反相色谱分离。相同长度的肽之间的保留时间差异随着肽链长度的增加而增大，对于两根14个残基的肽在C8柱上差异为2.9分钟，对于35个残基的肽最大差异为7.3分钟。从这两组肽在反相色谱中的观察到的和预测的保留时间，我们已经证明不仅可以预测两亲性α-螺旋在反相色谱中的保留行为，而且还可以根据肽的保留数据推断肽中两亲性α-螺旋结构的存在。如果从这样的研究中我们最终能够仅根据氨基酸序列信息预测肽与疏水基质结合时的二级结构，那么我们也许能够将这种预测能力外推到更大的多肽或蛋白质中相同序列的构象。
