Abstract：
Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2-6 °C in thermal inactivation temperature and no decrease in functionality.
摘要：
稳定蛋白质而不妨碍其功能是蛋白质工程和设计中的中心任务。PYR1是一种植物激素受体，已被设计为结合不同的小分子配体。我们寻找了一组广义突变,其将提供稳定性而不影响具有不同配体结合能力的PYR1变体的功能性。为此，我们使用了全局多突变分析(GMMA)方法，它可以识别具有稳定作用且不会降低功能的替代。GMMA具有发现在不同序列环境中稳定的取代的额外益处,并且我们假设将GMMA应用于具有不同功能的PYR1将鉴定这组广义突变。的确,对具有两种不同功能的PYR1变体的文库进行FACS和深度测序，并应用GMMA分析鉴定出5个取代，当插入到四个PYR1变体中时，每个变体都结合一个独特的配体，提供了2°C-6°C的热失活温度的增加和功能没有降低。
