Design of functional ferritin-like proteins with hydrophobic cavities.
Design of functional ferritin-like proteins with hydrophobic cavities.
复制标题
具有疏水空腔的功能性铁蛋白样蛋白的设计。
DOI：
10.1021/ja057069x
复制
发表时间：
2006
影响因子：
15
通讯作者：
Dmochowski,IvanJ
中科院分区：
文献类型：
--
作者：
Swift,Joe;Wehbi,WilliamA;Kelly,BrennaD;Stowell,XiaoranFu;Saven,JefferyG;Dmochowski,IvanJ
铁蛋白四螺旋束亚单位自组装，形成一个稳定的多聚体，具有一个大的中心亲水空腔，金属离子在那里结合。为了探索这种反应容器的多功能性，计算设计被用来在一个十二聚体铁蛋白样蛋白DPS内产生具有越来越多非极性表面积的空腔。空穴突变体，其中多达120个表面可达的亲水残基被疏水氨基酸取代，通过尺寸排除层析和动态光散射测量表明仍然可以正确组装。野生型DPS表现出高度协作的亚基折叠和组装，这通过色氨酸荧光和紫外圆二色谱的变化进行监测。疏水空腔突变体表现出明显的不合作去折叠行为，其中一个突变体在胍变性后形成部分组装的中间体。尽管DPS对这种变性的稳定性随着非极表面积的增加而降低，但所有蛋白质都表现出较高的熔融温度，Tm=74−90°C。尽管有大量突变，但仍保持了近天然的矿化铁能力。这项工作展示了铁蛋白支架的多功能性，用于设计具有新特性的大型蛋白质腔。
Ferritin four-helix bundle subunits self-assemble to create a stable multimer with a large central hydrophilic cavity where metal ions bind. To explore the versatility of this reaction vessel, computational design was used to generate cavities with increasingly apolar surface areas inside a dodecameric ferritin-like protein, Dps. Cavity mutants, in which as many as 120 surface accessible hydrophilic residues were replaced with hydrophobic amino acids, were shown to still assemble properly using size-exclusion chromatography and dynamic light scattering measurements. Wild-type Dps exhibited highly cooperative subunit folding and assembly, which was monitored by changes in Trp fluorescence and UV circular dichroism. The hydrophobic cavity mutants showed distinctly less cooperative unfolding behavior, with one mutant forming a partially assembled intermediate upon guanidine denaturation. Although the stability of Dps to such denaturation decreased with increasing apolar surface area, all proteins exhibited high melting temperatures,Tm= 74−90 °C. Despite the large number of mutations, near-native ability to mineralize iron was maintained. This work illustrates the versatility of the ferritin scaffold for engineering large protein cavities with novel properties.