Genetic sensors with unique combinations of DNA recognition and allosteric response can be created by hybridizing DNA-binding modules (DBMs) and ligand-binding modules (LBMs) from distinct transcriptional repressors. This module swapping approach is limited by incompatibility between DBMs and LBMs from different proteins, due to the loss of critical module-module interactions after hybridization. We determine a design strategy for restoring key interactions between DBMs and LBMs by using a computational model informed by coevolutionary traits in the LacI family. This model predicts the influence of proposed mutations on protein structure and function, quantifying the feasibility of each mutation for rescuing hybrid repressors. We accurately predict which hybrid repressors can be rescued by mutating residues to reinstall relevant module-module interactions. Experimental results confirm that dynamic ranges of gene expression induction were improved significantly in these mutants. This approach enhances the molecular and mechanistic understanding of LacI family proteins, and advances the ability to design modular genetic parts.

通过将来自不同转录抑制子的 DNA 结合模块 (DBM) 和配体结合模块 (LBM) 杂交，可以创建具有 DNA 识别和变构反应独特组合的遗传传感器。这种模块交换方法受到来自不同蛋白质的 DBM 和 LBM 之间不相容性的限制，这是由于杂交后关键模块-模块相互作用的丢失。我们确定了一种设计策略，通过使用由 LacI 家族的共同进化特征提供的计算模型来恢复 DBM 和 LBM 之间的关键相互作用。该模型预测了所提出的突变对蛋白质结构和功能的影响，量化了每个突变拯救混合阻遏物的可行性。我们准确预测哪些混合抑制子可以通过突变残基来重新安装相关的模块-模块相互作用来拯救。实验结果证实，这些突变体的基因表达诱导的动态范围显着改善。这种方法增强了对 LacI 家族蛋白的分子和机制理解，并提高了设计模块化遗传部分的能力。