Thermostability and Molecular Encapsulation Within an Engineered Caged Protein Scaffold
Thermostability and Molecular Encapsulation Within an Engineered Caged Protein Scaffold
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DOI：
10.1002/bit.21988
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发表时间：
2008-11-01
影响因子：
3.8
通讯作者：
Wang, Szu-Wen
中科院分区：
文献类型：
--
作者：
Dalmau, Merce;Lim, Sierin;Wang, Szu-Wen
自组装生物复合物，如病毒衣壳已被操纵，以发挥作用，在创新的纳米技术应用。来自嗜热脂肪芽孢杆菌的丙酮酸脱氢酶的E2组分形成十二面体复合物，并可能为这些目的提供另一个平台。在这项调查中，我们表明，这种蛋白质组装表现出不寻常的稳定性，并可以修改封装模型药物分子。为了将E2蛋白提取到其结构支架核心，我们合成了一个优化的截短基因，用于在大肠杆菌中表达。用动态光散射和透射电子显微镜证实了所得支架的正确组装和十二面体结构。使用圆二色性和差示扫描量热法，我们发现复合物的热稳定性异常高，展开起始温度为81.1 +/- 0.9 ℃，表观中点展开温度为91.4 +/- 1.4 ℃。为了评估这种支架用于包封客体分子的潜力，我们在残基381和239处制备了变体，其改变了中空内腔的物理化学性质。这些突变体在该空腔内产生60和120个突变，组装成正确的结构，并表现出与野生型支架相当的高热稳定性。为了显示该支架的适用性，将两种不同的荧光染料分子共价偶联至位点381处的半胱氨酸突变体。我们证明了这些突变可以引入非天然功能，并使分子封装在空腔内，同时仍然保留十二面体结构。这种支架的异常坚固的性质及其对内部变化的顺从性揭示了其在纳米级应用中的潜力。
Self-assembling biological complexes such as viral capsids have been manipulated to function in innovative nanotechnology applications. The E2 component of pyruvate dehydrogenase from Bacillus stearothermophilus forms a dodecahedral complex and potentially provides another platform for these purposes. In this investigation, we show that this protein assembly exhibits unusual stability and can be modified to encapsulate model drug molecules. To distill the E2 protein down to its structural scaffold core, we synthesized a truncated gene optimized for expression in Escherichia coli. The correct assembly and dodecahedral structure of the resulting scaffold was confirmed with dynamic light scattering and transmission electron microscopy. Using circular dichroism and differential scanning calorimetry, we found the thermostability of the complex to be unusually high, with an onset temperature of unfolding at 81.1 +/- 0.9 degrees C and an apparent midpoint unfolding temperature of 91.4 +/- 1.4 degrees C. To evaluate the potential of this scaffold for encapsulation of guest molecules, we made variants at residues 381 and 239 which altered the physicochemical properties of the hollow internal cavity. These mutants, yielding 60 and 120 mutations within this cavity, assembled into the correct architecture and exhibited high thermostability that was comparable to the wild-type scaffold. To show the applicability of this scaffold, two different fluorescent dye molecules were covalently coupled to the cysteine mutant at site 381. We demonstrate that these mutations can introduce non-native functionality and enable molecular encapsulation within the cavity while still retaining the dodecahedral structure. The unusually robust nature of this scaffold and its amenability to internal changes reveal its potential for nanoscale applications.