Mooers Blaine H M, Datta Deepshikha, Baase Walter A, Zollars Eric S, Mayo Stephen L, Matthews Brian W
Department of Physics, Institute of Molecular Biology, Howard Hughes Medical Institute, 1229 University of Oregon, Eugene, OR 97403-1229, USA.
J Mol Biol. 2003 Sep 19;332(3):741-56. doi: 10.1016/s0022-2836(03)00856-8.
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.
如程序ORBIT中所实现的自动蛋白质重新设计，被用于重新设计噬菌体T4溶菌酶的核心。在C端结构域中总共26个埋藏或部分埋藏的位点，其序列和侧链构象被允许变化，而主链和未选择的侧链保持固定。一个具有七个取代的变体（“Core-7”）被鉴定为具有最有利的能量。对蛋氨酸残基的存在设置惩罚后重复了重新设计实验。在这种情况下，重新设计的蛋白质（“Core-10”）有十个氨基酸变化。这两种设计的蛋白质，以及组成型单突变体和几个单点回复突变体，在大肠杆菌中过量表达、纯化，并进行晶体学和热分析。热力学和结构数据表明，虽然两种重新设计的蛋白质都没有比野生型蛋白质更稳定，但实现了一些重新包装。使用蛋氨酸惩罚被证明是有效的。Core-10预测结构中的几个侧链旋转异构体与观察到的不同。侧链不是转变为设计过程预测的新旋转异构体，而是倾向于保持与天然分子中相似的构象。相比之下，相对于设计结构和野生型，主链的部分变化高达2.8埃。溶菌酶分子内存在的水分子在设计过程中被去除。在重新设计的蛋白质中，产生的空洞在某种程度上被侧链原子重新占据。然而，在观察到的结构中，水分子仍然结合在其原始位置或附近。这表明在设计过程中保留这些水分子可能更可取。结果强调了典型蛋白质核心内发生的堆积的特异性。虽然核心内的点取代是可以容忍的，但它们几乎总是导致稳定性的丧失。同样，取代的组合也可能被容忍，但通常会使蛋白质不稳定。T4溶菌酶的经验表明，在没有主链移位的情况下，实现保留或增强稳定性的通用核心重新包装方法可能很困难。
