Exploring sequence/folding space:: Folding studies on multiple hydrophobic core mutants of ubiquitin
Exploring sequence/folding space:: Folding studies on multiple hydrophobic core mutants of ubiquitin
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DOI：
10.1021/bi0361620
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发表时间：
2004-05-11
期刊：
影响因子：
2.9
通讯作者：
Jackson, SE
中科院分区：
文献类型：
--
作者：
Benítez-Cardoza, CG;Stott, K;Jackson, SE
研究了泛素和两个多疏水核心突变体的稳定性、动力学和结构特性。其中一个突变体（U4）在疏水核心中具有七个取代（M1L、I3 L、V5 I、I3 F、L15 V）。V17 M和V26 L）。平均而言，它的侧链比野生型大，因此可以认为它具有过度堆积的核心。另一个突变体（U 7）具有两个取代（I3 V和I13 V）。平均而言，它具有比野生型更小的侧链，因此可以认为它是欠包装的。这三种蛋白质折叠良好，并显示出相似的骨架动力学（T-1，T-2和HNOE值），表明规则的二级结构在相同的残基范围内延伸。测定了U_4的晶体结构。最终的R因子和无R为0.198和0.248。分别在2.18埃分辨率下。U4的结构与野生型泛素非常相似。非常好C-α原子的位置沿整个主链沿着几乎没有变化，并保持氢键网络。疏水核心的突变通过突变和非突变残基核心中侧链的小移动来调节。展开和重折叠动力学研究表明，U4展开的速率最高，然而，其重折叠速率常数是非常相似的野生型蛋白质。相反，U 7似乎是最不稳定的蛋白质;其重折叠速率常数小于其他两种蛋白质。这是证实了停流技术和H/D交换方法。这项工作说明了重新包装小蛋白质的疏水核心的可能性，并在从头设计稳定的蛋白质具有重要意义。
The stability, dynamic, and structural properties of ubiquitin and two Multiple hydrophobic core mutants were studied. One of the mutants (U4) has seven substitutions in the hydrophobic core (M1L, I3L, V5I, I3F, L15V. V17M, and V26L). On average, its side chains are larger than the wild-type, and it can thus be thought of as having an overpacked core. The other mutant (U7) has two substitutions (I3V and I13V). On average, it has smaller side chains than the wild-type, and it can therefore be considered to be underpacked. The three proteins are well-folded and show similar backbone dynamics (T-1, T-2, and HNOE values), indicating that the regular secondary structure extends over the same residue ranges. The crystallographic structure of U4 was determined. The final R-factor and R-free are 0.198 and 0.248. respectively, at 2.18 Angstrom resolution. The structure of U4 is very similar to wild-type ubiquitin. Remarkably. there are almost no changes in the positions of the C-alpha atoms along the entire backbone, and the hydrogen-bonding network is maintained. The mutations of the hydrophobic core are accommodated by small movements of side chains in the core of mutated and nonmutated residues. Unfolding and refolding kinetic studies revealed that U4 unfolds with the highest rates; however, its refolding rate constants are very similar to those of the wild-type protein. Conversely, U7 seems to be the most destabilized protein; its refolding rate constant is smaller than the other two proteins. This was confirmed by stopped-flow techniques and by H/D exchange methodologies. This work illustrates the possibility of repacking the hydrophobic core of small proteins and has important implications in the de novo design of stable proteins.