Tanaka T, Kuroda Y, Kimura H, Kidokoro S, Nakamura H
Protein Engineering Research Institute, Osaka, Japan.
Protein Eng. 1994 Aug;7(8):969-76. doi: 10.1093/protein/7.8.969.
A de novo protein design has been made to understand the unique packing of natural proteins that have a beta/alpha-barrel fold. A carefully designed 207 amino acid sequence was synthesized using an Escherichia coli expression system and the structural and thermodynamic characteristics of the purified protein were studied. At neutral pH the protein is soluble and monomeric, with large amounts of secondary structure and a hydrophobic core, although the broad resonance peaks of its NMR spectrum suggest that the designed protein does not have a unique structure with tightly packed side chains. In an H-D exchange experiment, no amido protons of the designed protein exchanged slowly with deuterons. At acidic pH, thermal unfolding was observed with a remarkable change in the excess heat capacity measured directly by a differential scanning microcalorimeter. The enthalpy and entropy differences at 110 degrees C, extrapolated from analyzed thermodynamic parameters, are approximately 1/3 of the common values for natural proteins. These measurements indicate that the folding is significantly cooperative as expected, but that the protein is still loosely packed.
为了理解具有β/α-桶状折叠的天然蛋白质的独特堆积方式，进行了从头蛋白质设计。使用大肠杆菌表达系统合成了精心设计的207个氨基酸序列，并研究了纯化蛋白质的结构和热力学特性。在中性pH下，该蛋白质是可溶的且为单体，具有大量二级结构和疏水核心，尽管其核磁共振谱的宽共振峰表明设计的蛋白质没有侧链紧密堆积的独特结构。在氢-氘交换实验中，设计蛋白质的酰胺质子没有与氘缓慢交换。在酸性pH下，观察到热解折叠，通过差示扫描微量热法直接测量的过量热容有显著变化。从分析的热力学参数外推得到的110℃时的焓变和熵变约为天然蛋白质常见值的1/3。这些测量结果表明，折叠如预期的那样具有显著的协同性，但该蛋白质仍然堆积松散。
