Swift Joe, Wehbi William A, Kelly Brenna D, Stowell Xiaoran Fu, Saven Jeffery G, Dmochowski Ivan J
Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, USA.
J Am Chem Soc. 2006 May 24;128(20):6611-9. doi: 10.1021/ja057069x.
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, T(m) = 74-90 degrees 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.
铁蛋白四螺旋束亚基自组装形成一个稳定的多聚体，其具有一个大的中央亲水性空腔，金属离子可在其中结合。为了探索这个反应容器的多功能性，采用计算设计在十二聚体铁蛋白样蛋白Dps内部生成具有越来越大非极性表面积的空腔。通过尺寸排阻色谱和动态光散射测量表明，多达120个表面可及的亲水性残基被疏水性氨基酸取代的空腔突变体仍能正确组装。野生型Dps表现出高度协同的亚基折叠和组装，通过色氨酸荧光和紫外圆二色性的变化进行监测。疏水性空腔突变体表现出明显较少的协同解折叠行为，其中一个突变体在胍变性时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性表面积的增加而降低，但所有蛋白质都表现出高熔点温度，Tm = 74 - 90℃。尽管有大量突变，但仍保持了接近天然的铁矿化能力。这项工作说明了铁蛋白支架在工程化具有新特性的大蛋白空腔方面的多功能性。
