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首页> 外文期刊>Journal of Molecular Biology >Repacking the Core of T4 lysozyme by automated design.
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Repacking the Core of T4 lysozyme by automated design.

机译：通过自动设计重新包装T4溶菌酶的核心。

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Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective.Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type.Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.
机译：按照ORBIT程序实现的自动化蛋白质重新设计用于重新设计噬菌体T4溶菌酶的核心。 C-末端结构域中总共26个掩埋或部分掩埋的位点被允许改变其序列和侧链构象，而主链和未选择的侧链保持固定。具有七个取代的变体（“ Core-7”）被鉴定为具有最有利的能量。重复进行重新设计实验，但要保留蛋氨酸残基。在这种情况下，重新设计的蛋白质（“ Core-10”）具有10个氨基酸变化。在大肠杆菌中过表达了两个设计的蛋白质以及组成的单个突变体和几个单点回复体，进行了纯化，并进行了晶体学和热分析。热力学和结构数据表明，尽管没有一种重新设计的蛋白质比野生型蛋白质更稳定，但还是实现了一些重新包装。蛋氨酸惩罚的使用被证明是有效的。Core-10预测结构中的几个侧链旋转异构体与观察到的不同。侧链倾向于保持与天然分子相似的构象，而不是改变设计过程中预测的新旋转异构体。相比之下，相对于设计的结构和野生型，骨干部分最多可改变2.8A。在设计过程中，溶菌酶分子内存在的水分子被去除了。在重新设计的蛋白质中，所得的空洞在某种程度上被侧链原子所占据。然而，在观察到的结构中，水分子仍结合在其原始位置或附近。这表明在设计过程中最好将此类水分子留在原地。结果强调了典型蛋白质核心内包装的特异性。尽管可以容忍核心内的点替换，但它们几乎总是导致稳定性下降。同样，取代的组合也可以被接受，但通常会使蛋白质不稳定。 T4溶菌酶的经验表明，如果不提供主干的转换，可能很难实现具有保留或增强稳定性的一般核心重组方法。

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