Relative influence of hydrophobicity and net charge in the aggregation of two homologous proteins
Relative influence of hydrophobicity and net charge in the aggregation of two homologous proteins
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DOI：
10.1021/bi030135s
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发表时间：
2003-12-30
期刊：
影响因子：
2.9
通讯作者：
Chiti, F
中科院分区：
文献类型：
--
作者：
Calamai, M;Taddei, N;Chiti, F
一个潜在的淀粉样蛋白必须至少部分解开才能形成淀粉样聚集体。然而，蛋白质部分或完全展开状态的聚集至少受其他三个因素的调节：疏水性、形成二级结构的倾向和多肽链的净电荷。我们建议评估净电荷相对于其他因素对蛋白质聚集和淀粉样变性的相对重要性。为此，我们使用了两种先前被证明能够在体外形成淀粉样纤维的同源蛋白，来自大肠杆菌的HypF的N-末端结构域(HypF-N)和人肌肉酰基磷酸酶(ACP)。HypF-N从部分展开的构象集合的聚集过程比ACP快约1000倍。这主要是由于HypF-N的疏水性较高，净电荷较低。利用蛋白质工程方法，我们将ACP的净电荷降低到与野生型HypF-N相同的值，将HypF-N的净电荷增加到与野生型ACP相同的值。氨基酸取代被选择来最小化疏水性和二级结构倾向的变化。我们能够估计，这两个野生型蛋白之间的净电荷差异贡献了它们聚集率差异的20%-25%。了解这些作用力在蛋白质聚集中的相对影响对于阐明聚集过程的复杂性、预测自然突变的影响以及准确的蛋白质设计具有重要意义。
A potentially amyloidogenic protein has to be at least partially unfolded to form amyloid aggregates. However, aggregation of the partially or totally unfolded state of a protein is modulated by at least three other factors: hydrophobicity, propensity to form secondary structure, and net charge of the polypeptide chain. We propose to evaluate the relative importance of net charge, as opposed to the other factors, on protein aggregation and amyloidogenicity. For this aim, we have used two homologous proteins that were previously shown to be able to form amyloid fibrils in vitro, the N-terminal domain of HypF from Escherichia coli (HypF-N) and human muscle acylphosphatase (AcP). The aggregation process from an ensemble of partially unfolded conformations is ca. 1000-fold faster for HypF-N than for AcP. This difference can mainly be attributed to a higher hydrophobicity and a lower net charge for HypF-N than for AcP. By using protein engineering methods, we have decreased the net charge of AcP to a value identical to that of wild-type HypF-N and increased the net charge of HypF-N to a value identical to that of wild-type AcP. Amino acid substitutions were selected to minimize changes in hydrophobicity and secondary structure propensities. We were able to estimate that the difference in net charge between the two wild-type proteins contributes to 20-25% of the difference in their aggregation rates. An understanding of the relative influences of these forces in protein aggregation has implications for elucidating the complexity of the aggregation process, for predicting the effect of natural mutations, and for accurate protein design.