Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas 78712, United States.
Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States.
J Am Chem Soc. 2020 Dec 9;142(49):20796-20805. doi: 10.1021/jacs.0c09802. Epub 2020 Nov 25.
Cellular membranes are densely covered by proteins. Steric pressure generated by protein collisions plays a significant role in shaping and curving biological membranes. However, no method currently exists for measuring steric pressure at membrane surfaces. Here, we developed a sensor based on Förster resonance energy transfer (FRET), which uses the principles of polymer physics to precisely detect changes in steric pressure. The sensor consists of a polyethylene glycol chain tethered to the membrane surface. The polymer has a donor fluorophore at its free end, such that FRET with acceptor fluorophores in the membrane provides a real-time readout of polymer extension. As a demonstration of the sensor, we measured the steric pressure generated by a model protein involved in membrane bending, the N-terminal homology domain (ENTH) of Epsin1. As the membrane becomes crowded by ENTH proteins, the polymer chain extends, increasing the fluorescence lifetime of the donor. Drawing on polymer theory, we use this change in lifetime to calculate steric pressure as a function of membrane coverage by ENTH, validating theoretical equations of state. Further, we find that ENTH's ability to break up larger vesicles into smaller ones correlates with steric pressure rather than the chemistry used to attach ENTH to the membrane surface. This result addresses a long-standing question about the molecular mechanisms of membrane remodeling. More broadly, this sensor makes it possible to measure steric pressure in situ during diverse biochemical events that occur on membrane surfaces, such as membrane remodeling, ligand-receptor binding, assembly of protein complexes, and changes in membrane organization.
细胞膜表面密布着蛋白质。由蛋白质碰撞产生的空间位阻压力在塑造和弯曲生物膜方面起着重要作用。然而，目前还没有测量膜表面位阻压力的方法。在这里，我们开发了一种基于Förster 共振能量转移（FRET）的传感器，该传感器利用聚合物物理原理精确地检测位阻压力的变化。该传感器由一个连接在膜表面的聚乙二醇链组成。该聚合物在其自由端具有供体荧光团，因此与膜中的受体荧光团发生 FRET 可实时读取聚合物的延伸。作为传感器的演示，我们测量了参与膜弯曲的模型蛋白，即 Epsin1 的 N 端同源结构域（ENTH）产生的位阻压力。随着膜被 ENTH 蛋白占据，聚合物链延伸，增加了供体的荧光寿命。根据聚合物理论，我们利用寿命的这种变化来计算位阻压力作为 ENTH 覆盖膜的函数，验证了状态方程的理论公式。此外，我们发现 ENTH 能够将较大的囊泡分裂成较小的囊泡的能力与位阻压力相关，而不是与将 ENTH 连接到膜表面的化学物质相关。这一结果解决了关于膜重塑分子机制的一个长期存在的问题。更广泛地说，这种传感器使得在膜表面发生的各种生化事件（如膜重塑、配体-受体结合、蛋白质复合物组装和膜组织变化）中能够原位测量位阻压力。
