Contribution of individual side-chains to the stability of BPTI examined by alanine-scanning mutagenesis.
Contribution of individual side-chains to the stability of BPTI examined by alanine-scanning mutagenesis.
复制标题
通过丙氨酸扫描诱变检查各个侧链对 BPTI 稳定性的贡献。
DOI：
10.1006/jmbi.1995.0304
复制
发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Kim,PS
中科院分区：
文献类型：
--
作者：
Yu,MH;Weissman,JS;Kim,PS
牛胰蛋白酶抑制剂（BPTI）作为一种重要的模型系统，几乎可以检测蛋白质结构的各个方面。然而，突变对BPTI热力学稳定性影响的系统研究受到蛋白质极端稳定性的限制。BPTI的衍生物仅含有5-55二硫键，称为[5-55]Ala，先前已被证明折叠成与天然BPTI非常相似的结构，并且是一种功能性胰蛋白酶抑制剂。[5-55]在39°C的熔融温度下，alaau经历了可逆的热展开转变，因此非常适合进行稳定性研究。使用丙氨酸扫描诱变方法，我们研究了BPTI[5-55]衍生物中每个侧链对稳定性的贡献。这些研究证明了两个主要由芳香残基簇组成的疏水核以及内部氢键网络在稳定BPTI中的重要性。总的来说，埋藏表面积的变化与极性和疏水残基的稳定性之间存在很强的关系，比例常数分别为50和20 cal/Å2。所有的丙氨酸取代都没有稳定亚拉氨酸[5-55]。尽管如此，大约60%（28/46）的丙氨酸突变体在不到10°C的温度下不稳定，这表明一种含有多达一半残基是丙氨酸的BPTI形式可以折叠成类似于天然结构的稳定结构。
Bovine pancreatic trypsin inhibitor (BPTI) serves as an important model system for the examination of almost all aspects of protein structure. Systematic studies of the effects of mutation on the thermodynamic stability of BPTI, however, have been limited by the extreme stability of the protein. A derivative of BPTI containing only the 5–55 disulfide bond, termed [5–55]Ala, has been shown previously to fold into a structure very similar to that of native BPTI and to be a functional trypsin inhibitor. [5–55]Alaundergoes a reversible thermal unfolding transition with a melting temperature of 39°C, and is therefore well suited for stability studies. Using an alanine-scanning mutagenesis approach, we have examined the contribution to stability of each side-chain in the [5–55]Aladerivative of BPTI. These studies demonstrate the importance of the two hydrophobic cores composed largely of clusters of aromatic residues, as well as the internal hydrogen-bonding network, in stabilizing BPTI. Overall, there is a strong relationship between change in buried surface area and stability for both polar and hydrophobic residues, with proportionality constants of 50 and 20 cal/Å2, respectively. None of the alanine substitutions substantially stabilized [5–55]Ala. Nonetheless, approximately 60% (28/46) of the alanine mutants were destabilized by less than 10°C, suggesting that a form of BPTI with up to half of its residues being alanine could fold into a stable structure resembling the native one.