Kuroda Y, Kim P S
Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research Department of Biology, MIT, Cambridge, MA, 02142, USA.
J Mol Biol. 2000 May 5;298(3):493-501. doi: 10.1006/jmbi.2000.3622.
Recent studies indicate that a fraction of the information contained in an amino acid sequence may be sufficient for specifying a native protein structure. An earlier alanine-scanning experiment conducted on bovine pancreatic trypsin inhibitor (BPTI; 58 residues) suggested that if cumulative mutations have additive effects on protein stability, a native protein structure could be built from BPTI sequences that contained many alanine residues distributed throughout the protein. To test this hypothesis, we designed and produced six BPTI mutants containing from 21 to 29 alanine residues. We found that the melting temperature of mutants containing up to 27 alanine residues (48 % of the total number of residues) could be predicted quite well by the sum of the change in melting temperature for the single mutations. Additionally, these same mutants folded into a native-like structure, as judged by their cooperative thermal denaturation curves and heteronuclear multiple quantum correlation (HMQC) NMR spectra. A BPTI mutant containing 22 alanine residues was further shown by 2D and 3D-NMR to fold into a structure very similar to that of native BPTI, and to be a functional trypsin inhibitor. These results provide insight into the extent to which native protein structure and function can be achieved with a highly simplified amino acid sequence.
最近的研究表明，氨基酸序列中包含的一部分信息可能足以确定天然蛋白质的结构。早期对牛胰蛋白酶抑制剂（BPTI；58个残基）进行的丙氨酸扫描实验表明，如果累积突变对蛋白质稳定性有累加效应，那么天然蛋白质结构可以由在整个蛋白质中分布有许多丙氨酸残基的BPTI序列构建而成。为了验证这一假设，我们设计并制备了六个含有21至29个丙氨酸残基的BPTI突变体。我们发现，含有多达27个丙氨酸残基（占残基总数的48%）的突变体的解链温度可以通过单个突变的解链温度变化之和很好地预测。此外，从它们的协同热变性曲线和异核多量子相关（HMQC）核磁共振谱判断，这些相同的突变体折叠成了类似天然的结构。一个含有22个丙氨酸残基的BPTI突变体通过二维和三维核磁共振进一步表明，它折叠成了一种与天然BPTI非常相似的结构，并且是一种功能性胰蛋白酶抑制剂。这些结果为深入了解用高度简化的氨基酸序列能够实现天然蛋白质结构和功能的程度提供了线索。
