The redesign of a macromolecular binding interface and corresponding alteration of recognition specificity is a challenging endeavor that remains recalcitrant to computational approaches. This is particularly true for the redesign of DNA binding specificity, which is highly dependent upon bending, hydrogen bonds, electrostatic contacts, and the presence of solvent and counterions throughout the molecular interface. Thus, redesign of protein-DNA binding specificity generally requires iterative rounds of amino acid randomization coupled to selections. Here, we describe the importance of scaffold thermostability for protein engineering, coupled with a strategy that exploits the protein's specificity profile, to redesign the specificity of a pair of meganucleases toward three separate genomic targets. We determine and describe a series of changes in protein sequence, stability, structure, and activity that accumulate during the engineering process, culminating in fully retargeted endonucleases.

大分子结合界面的重新设计和识别特异性的相应改变是一项具有挑战性的努力，对计算方法仍然顽固。对于 DNA 结合特异性的重新设计尤其如此，它高度依赖于弯曲、氢键、静电接触以及整个分子界面中溶剂和抗衡离子的存在。因此，蛋白质-DNA 结合特异性的重新设计通常需要与选择相结合的氨基酸随机化迭代轮。在这里，我们描述了支架热稳定性对于蛋白质工程的重要性，以及利用蛋白质特异性特征的策略，以重新设计一对大范围核酸酶对三个独立基因组靶标的特异性。