A Förster Resonance Energy Transfer-Based Sensor of Steric Pressure on Membrane Surfaces.
A Förster Resonance Energy Transfer-Based Sensor of Steric Pressure on Membrane Surfaces.
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基于福斯特共振能量转移的膜表面空间压力传感器。
DOI：
10.1021/jacs.0c09802
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发表时间：
2020-12-09
影响因子：
15
通讯作者：
Stachowiak JC
中科院分区：
文献类型：
--
作者：
Houser JR;Hayden CC;Thirumalai D;Stachowiak JC
细胞膜被蛋白质致密地覆盖。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，目前还没有用于测量膜表面的空间压力的方法。在这里，我们开发了一种基于Förster共振能量转移（FRET）的传感器，它使用聚合物物理学的原理来精确检测空间压力的变化。该传感器由拴在膜表面的聚乙二醇链组成。聚合物在其自由端具有供体荧光团，使得具有膜中的受体荧光团的FRET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质（Epsin1的ENTH结构域）产生的空间压力。随着ENTH蛋白质使膜变得拥挤，聚合物链延伸，增加供体的荧光寿命。利用聚合物理论，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，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，而不是用于将ENTH附着到膜表面的化学物质。这一结果解决了一个长期存在的问题，膜重塑的分子机制。更广泛地说，这种传感器可以在膜表面发生的各种生物化学事件（如膜重塑、配体-受体结合、蛋白质复合物组装和膜组织变化）期间原位测量空间压力。
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 develop 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 ENTH domain 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 breakup 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.
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影响因子：
11.8
作者：
Kory N;Thiam AR;Farese RV Jr;Walther TC
通讯作者：
Walther TC
影响因子：
15
作者：
Jiang Z;de Messieres M;Lee JC
通讯作者：
Lee JC
影响因子：
21.3
作者：
通讯作者：
--
DOI：
10.1073/pnas.1616199114
发表时间：
2017-04-18
影响因子：
11.1
作者：
Snead, Wilton T.;Hayden, Carl C.;Stachowiak, Jeanne C.
通讯作者：
Stachowiak, Jeanne C.
影响因子：
4.4
作者：
CARNAHAN, NF;STARLING, KE
通讯作者：
STARLING, KE