Lu Stephen M, Hodges Robert S
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Protein Sci. 2004 Mar;13(3):714-26. doi: 10.1110/ps.03443204.
The alpha-helical coiled-coil motif is characterized by a heptad repeat pattern (abcdefg)(n) in which residues a and d form the hydrophobic core. Long coiled-coils (e.g., tropomyosin, 284 residues per polypeptide chain) typically do not have a continuous hydrophobic core of stabilizing residues, but rather one that consists of alternating clusters of stabilizing and destabilizing residues. We have arbitrarily defined a cluster as a minimum of three consecutive stabilizing or destabilizing residues in the hydrophobic core. We report here on a series of two-stranded, disulfide-bridged parallel alpha-helical coiled-coils that contain a central cassette of three consecutive hydrophobic core positions (d, a, and d) with a destabilizing cluster of three consecutive Ala residues in the hydrophobic core on each side of the cassette. The effect of adding one to three stabilizing hydrophobes in these positions (Leu or Ile; denoted as [see text]) was investigated. Alanine residues (denoted as [see text]) are used to represent destabilizing residues. The peptide with three Ala residues in the d a d cassette positions ([see text]) was among the least stable coiled-coil (T(m) = 39.3 degrees C and Urea(1/2) = 1.9 M). Surprisingly, the addition of one stabilizing hydrophobe (Leu) to the cassette or two stabilizing hydrophobes (Leu), still interspersed by an Ala in the cassette ([see text]), also did not lead to any gain in stability. However, peptides with two adjacent hydrophobes in the cassette ([see text])([see text]) did show a gain in stability of 0.9 kcal/mole over the peptide with two interspersed hydrophobes ([see text]). Because the latter three peptides have the same inherent hydrophobicity, the juxtaposition of stabilizing hydrophobes leads to a synergistic effect, and thus a clustering effect. The addition of a third stabilizing hydrophobe to the cassette ([see text]) resulted in a further synergistic gain in stability of 1.7 kcal/mole (T(m) = 54.1 degrees C and Urea(1/2) = 3.3M). Therefore, the role of hydrophobicity in the hydrophobic core of coiled-coils is extremely context dependent and clustering is an important aspect of protein folding and stability.
α-螺旋卷曲螺旋基序的特征是七肽重复模式(abcdefg)(n)，其中a和d残基形成疏水核心。长的卷曲螺旋（例如，原肌球蛋白，每条多肽链有284个残基）通常没有由稳定残基组成的连续疏水核心，而是由稳定和不稳定残基交替簇组成的疏水核心。我们将一个簇任意定义为疏水核心中至少三个连续的稳定或不稳定残基。我们在此报告了一系列双链、二硫键桥连的平行α-螺旋卷曲螺旋，它们包含一个由三个连续疏水核心位置（d、a和d）组成的中央盒式结构，在盒式结构两侧的疏水核心中有一个由三个连续丙氨酸残基组成的不稳定簇。研究了在这些位置添加一到三个稳定疏水基团（亮氨酸或异亮氨酸；表示为[见正文]）的效果。丙氨酸残基（表示为[见正文]）用于表示不稳定残基。在d a d盒式结构位置有三个丙氨酸残基的肽（[见正文]）是最不稳定的卷曲螺旋之一（T(m)=39.3℃，尿素(1/2)=1.9M）。令人惊讶的是，在盒式结构中添加一个稳定疏水基团（亮氨酸）或两个稳定疏水基团（亮氨酸）（盒式结构中仍穿插一个丙氨酸）（[见正文]），也没有导致稳定性的任何增加。然而，在盒式结构中有两个相邻疏水基团的肽（[见正文]）（[见正文]）比有两个穿插疏水基团的肽（[见正文]）稳定性增加了0.9千卡/摩尔。因为后三种肽具有相同的固有疏水性，稳定疏水基团的并置导致协同效应，从而产生簇集效应。在盒式结构中添加第三个稳定疏水基团（[见正文]）导致稳定性进一步协同增加1.7千卡/摩尔（T(m)=54.1℃，尿素(1/2)=3.3M）。因此，疏水性在卷曲螺旋疏水核心中的作用极其依赖于上下文，簇集是蛋白质折叠和稳定性的一个重要方面。
