The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain
The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain
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DOI：
10.1110/ps.22202
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发表时间：
2002-03-01
期刊：
影响因子：
8
通讯作者：
McKnight, CJ
中科院分区：
文献类型：
--
作者：
Frank, BS;Vardar, D;McKnight, CJ
小型自主折叠蛋白质作为研究蛋白质折叠的模型系统很有意义，因为同一分子可用于实验和计算方法。问题仍然是这些最小化的肽模型系统如何代表较大的天然蛋白质。例如，一个最小化的蛋白质的核心是否像较大的蛋白质一样能够耐受突变？此外，最小化的蛋白质是否使用特殊的策略来指定和稳定其折叠结构？在这里，我们研究这些问题的35个残基自主折叠绒毛头部亚结构域（VHP亚结构域）。具体而言，我们专注于一组三个保守的苯丙氨酸（F）残基F47，F51和F58，形成大部分的疏水核心。这三个残基的方向，使它们可以提供稳定的芳香族-芳香族相互作用，这可能是至关重要的指定折叠。圆二色谱和1D-NMR光谱表明，单独取代这三个残基与亮氨酸的点突变是不稳定的，但保留了天然的VHP亚结构域折叠。在成对替换中，保留F58的双突变体可以采用天然折叠，而缺乏F58的两个双突变体不能。保留F58的双突变体的折叠表明芳香族簇内的芳香族-芳香族相互作用对于指定VHP亚结构域折叠不是必需的。VHP亚结构域在其疏水核心内耐受突变的能力表明，指定三维结构的信息分布在整个序列中，如在较大的蛋白质中所观察到的。因此，VHP亚结构域是更大的天然蛋白质的合理模型。
Small autonomously folding proteins are of interest as model systems to study protein folding, as the same molecule can be used for both experimental and computational approaches. The question remains as to how well these minimized peptide model systems represent larger native proteins. For example, is the core of a minimized protein tolerant to mutation like larger proteins are? Also, do minimized proteins use special strategies for specifying and stabilizing their folded structure? Here we examine these questions in the 35-residue autonomously folding villin headpiece subdomain (VHP subdomain). Specifically, we focus on a cluster of three conserved phenylalanine (F) residues F47, F51, and F58, that form most of the hydrophobic core. These three residues are oriented such that they may provide stabilizing aromatic-aromatic interactions that could be critical for specifying the fold. Circular dichroism and 1D-NMR spectroscopy show that point mutations that individually replace any of these three residues with leucine were destabilized, but retained the native VHP subdomain fold. In pair-wise replacements, the double mutant that retains F58 can adopt the native fold, while the two double mutants that lack F58 cannot. The folding of the double mutant that retains F58 demonstrates that aromatic-aromatic interactions within the aromatic cluster are not essential for specifying the VHP subdomain fold. The ability of the VHP subdomain to tolerate mutations within its hydrophobic core indicates that the information specifying the three dimensional structure is distributed throughout the sequence, as observed in larger proteins. Thus, the VHP subdomain is a legitimate model for larger, native proteins.