穿孔素（perforin）是一种形成孔的蛋白质，通过清除受病毒感染或肿瘤的细胞在免疫系统中起着至关重要的作用。它由免疫细胞的细胞毒性颗粒释放，并在靶脂质膜中形成孔道，以传递诱导细胞凋亡的蛋白酶。它是一种非常细胞毒的蛋白质，因此适应于不在产生细胞中起作用。其活性受到钙离子最佳活性所需的调控。然而，穿孔素对钙离子的确切亲和力尚未确定。我们进行了在存在或不存钙离子的情况下的分子动力学模拟，结果显示，至少需要结合三个钙离子才能使穿孔素稳定结合到脂质膜上。然后，我们采用表面等离子共振和微观尺度热致测流技术进行了生物物理学研究，估计了天然人源穿孔素和重组小鼠穿孔素对钙离子的结合亲和力。两种方法均显示小鼠穿孔素对钙离子具有几倍于人源穿孔素的结合亲和力。这被归因于小鼠穿孔素在位置488处的特殊残基色氨酸，而人源穿孔素在该位置上被精氨酸取代。这代表了一种额外的机制来控制人源穿孔素的活性。 版权所有 2023 Naneh, Kozorog, Merzel, Gilbert and Anderluh。

Perforin is a pore-forming protein that plays a crucial role in the immune system by clearing virus-infected or tumor cells. It is released from cytotoxic granules of immune cells and forms pores in targeted lipid membranes to deliver apoptosis-inducing granzymes. It is a very cytotoxic protein and is therefore adapted not to act in producing cells. Its activity is regulated by the requirement for calcium ions for optimal activity. However, the exact affinity of perforin for calcium ions has not yet been determined. We conducted a molecular dynamics simulation in the absence or presence of calcium ions that showed that binding of at least three calcium ions is required for stable perforin binding to the lipid membrane. Biophysical studies using surface plasmon resonance and microscale thermophoresis were then performed to estimate the binding affinities of native human and recombinant mouse perforin for calcium ions. Both approaches showed that mouse perforin has a several fold higher affinity for calcium ions than that of human perforin. This was attributed to a particular residue, tryptophan at position 488 in mouse perforin, which is replaced by arginine in human perforin. This represents an additional mechanism to control the activity of human perforin.Copyright © 2023 Naneh, Kozorog, Merzel, Gilbert and Anderluh.