Department of Biological Chemistry, Hebrew University of Jerusalem, Givat Ram Campus, 91906, Jerusalem, Israel.
Avram and Stella Goldstein-Goren Department of Biotechnology Engineering and the National Institute of Biotechnology, Ben-Gurion University of the Negev, P.O.B. 653, 8410501, Beer-Sheva, Israel.
Nat Commun. 2020 Jan 15;11(1):297. doi: 10.1038/s41467-019-13895-8.
Quantifying the effects of various mutations on binding free energy is crucial for understanding the evolution of protein-protein interactions and would greatly facilitate protein engineering studies. Yet, measuring changes in binding free energy (ΔΔG) remains a tedious task that requires expression of each mutant, its purification, and affinity measurements. We developed an attractive approach that allows us to quantify ΔΔG for thousands of protein mutants in one experiment. Our protocol combines protein randomization, Yeast Surface Display technology, deep sequencing, and a few experimental ΔΔG data points on purified proteins to generate ΔΔG values for the remaining numerous mutants of the same protein complex. Using this methodology, we comprehensively map the single-mutant binding landscape of one of the highest-affinity interaction between BPTI and Bovine Trypsin (BT). We show that ΔΔG for this interaction could be quantified with high accuracy over the range of 12 kcal mol displayed by various BPTI single mutants.
量化各种突变对结合自由能的影响对于理解蛋白质-蛋白质相互作用的进化至关重要，并且将极大地促进蛋白质工程研究。然而，测量结合自由能的变化（ΔΔG）仍然是一项繁琐的任务，需要表达每个突变体、对其进行纯化并进行亲和力测量。我们开发了一种有吸引力的方法，可在一次实验中对数千种蛋白质突变体进行量化ΔΔG。我们的方案将蛋白质随机化、酵母表面展示技术、深度测序以及少量有关纯化蛋白质的实验ΔΔG 数据点相结合，为同一蛋白质复合物的其余大量突变体生成ΔΔG 值。使用这种方法，我们全面绘制了 BPTI 和牛胰蛋白酶（BT）之间亲和力最高的相互作用之一的单突变体结合图谱。我们表明，对于该相互作用，在各种 BPTI 单突变体所表现出的 12kcal/mol 范围内，可以高精度地量化ΔΔG。
