A Förster Resonance Energy Transfer-Based Sensor of Steric Pressure on Membrane Surfaces,Journal of the American Chemical Society

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A Förster Resonance Energy Transfer-Based Sensor of Steric Pressure on Membrane Surfaces
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2020-11-25 , DOI: 10.1021/jacs.0c09802
Justin R Houser ₁ , Carl C Hayden ₁ , D Thirumalai ₂ , Jeanne C Stachowiak _{1,

3}

Affiliation

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.

中文翻译：

基于福斯特共振能量转移的膜表面空间压力传感器

细胞膜被蛋白质紧密覆盖。蛋白质碰撞产生的空间压力在生物膜的成形和弯曲中起着重要作用。然而，目前不存在测量膜表面空间压力的方法。在这里，我们开发了一种基于福斯特共振能量转移（FRET）的传感器，它利用聚合物物理原理来精确检测空间压力的变化。该传感器由连接到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，因此膜中带有受体荧光团的 FRET 可以提供聚合物延伸的实时读数。作为传感器的演示，我们测量了参与膜弯曲的模型蛋白（Epsin1 的 N 端同源域 (ENTH)）产生的空间压力。当膜被 ENTH 蛋白挤满时，聚合物链就会延伸，从而增加供体的荧光寿命。借鉴聚合物理论，我们利用寿命的这种变化来计算空间压力作为 ENTH 膜覆盖率的函数，从而验证理论状态方程。此外，我们发现 ENTH 将较大囊泡分解成较小囊泡的能力与空间压力相关，而不是与用于将 ENTH 附着到膜表面的化学物质相关。这一结果解决了有关膜重塑分子机制的长期存在的问题。更广泛地说，该传感器可以在膜表面发生的各种生化事件（例如膜重塑、配体-受体结合、蛋白质复合物组装和膜组织变化）期间原位测量空间压力。

更新日期：2020-11-25

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