Improvements in genetic code expansion have made preparing proteins with diverse functional groups almost routine. Nonetheless, unnatural amino acids (Uaas) pose theoretical burdens on protein solubility, and determinants of position-specific tolerability to Uaas remain underexplored. To broadly examine associations, we systematically assessed the effect of substituting the fluorescent Uaa, acridonylalanine, at more than 50 chemically, evolutionarily, and structurally diverse residues in two bacterial proteins: LexA and RecA. Surprisingly, properties that ostensibly contribute to Uaa tolerability-such as conservation, hydrophobicity, or accessibility-demonstrated no consistent correlations with resulting protein solubility. Instead, solubility is closely dependent on the location of the substitution within the overall tertiary structure, suggesting that intrinsic properties of protein domains, and not individual positions, are stronger determinants of Uaa tolerability. Consequently, those who seek to install Uaas in new target proteins should consider broadening, rather than narrowing, the types of residues screened for Uaa incorporation.

中文翻译：

---

使用荧光非天然氨基酸对可溶性蛋白质表达的系统评估显示没有可靠的耐受性预测指标。

遗传密码扩展的改进使得制备具有不同官能团的蛋白质几乎成为常规。尽管如此，非天然氨基酸 (Uaas) 对蛋白质溶解度造成了理论上的负担，并且对 Uaas 的位置特异性耐受性的决定因素仍未得到充分探索。为了广泛检查关联，我们系统地评估了在两种细菌蛋白 LexA 和 RecA 中的 50 多个化学、进化和结构多样化的残基处取代荧光 Uaa、吖啶酰丙氨酸的效果。令人惊讶的是，表面上有助于 Uaa 耐受性的特性（例如保守性、疏水性或可及性）表明与所得蛋白质溶解度没有一致的相关性。相反，溶解度与整个三级结构中的取代位置密切相关，表明蛋白质结构域的内在特性，而不是单个位置，是 Uaa 耐受性的更强决定因素。因此，那些寻求在新靶蛋白中安装 Uaas 的人应该考虑扩大而不是缩小为 Uaa 掺入筛选的残基类型。