Exploring homo-FRET to quantify the oligomer stoichiometry of membrane-bound proteins involved in a cooperative partition equilibrium.
Exploring homo-FRET to quantify the oligomer stoichiometry of membrane-bound proteins involved in a cooperative partition equilibrium.
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探索同型 FRET 来量化参与协同分配平衡的膜结合蛋白的寡聚物化学计量。
DOI：
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
A. Coutinho
中科院分区：
文献类型：
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作者：
A. Melo;A. Fedorov;M. Prieto;A. Coutinho
膜结合蛋白之间蛋白质-蛋白质相互作用的建立与几种生物学功能和功能障碍有关。在这里，提出了一个分析框架，使用能量homo转移直接探测定量的寡聚化状态的膜结合蛋白质从事三态合作分区。简单地说，该模型假设单体蛋白质分子分配到双层表面，并可逆地组装成具有k亚基的寡聚体。通过明确考虑含有i-标记单体的混合低聚物的各向异性与每个低聚物的标记亚基的数量成反比（Runnels和Scarlata极限），推导出将样品的整体稳态荧光各向异性与其分数标记相关的一般方程。该方法在描述Alexa Fluor 488荧光标记的溶菌酶（Lz-A488）与含磷脂酰丝氨酸的膜的静电相互作用方面非常稳健。在Lz-A488的荧光各向异性中检测到的显著降低总是与系统达到蛋白质的高膜表面密度（在低脂质与蛋白质（L/P）摩尔比下）相关。通过测量Lz-A488在这些条件下的各向异性衰减，进一步证实了能量均质转移诱导的荧光去偏振的发生。对在广泛的实验条件（脂质体的不同阴离子脂质含量、L/P摩尔比和蛋白质分数标记）下获得的稳态各向异性数据的全局分析证实，膜结合Lz-A488组装成低聚复合物，可能具有化学计量k = 6 ± 1。这项研究表明，即使在存在耦合的分配寡聚化平衡，稳态各向异性测量提供了一个简单而可靠的工具来监测膜结合蛋白的自组装。
The establishment of protein-protein interactions between membrane-bound proteins is associated with several biological functions and dysfunctions. Here, an analytical framework that uses energy homo transfer to directly probe quantitatively the oligomerization state of membrane-bound proteins engaged in a three-state cooperative partition is presented. Briefly, this model assumes that monomeric protein molecules partition into the bilayer surface and reversibly assemble into oligomers with k subunits. A general equation relating the overall steady-state fluorescence anisotropy of the sample to its fractional labeling was derived by considering explicitly that the anisotropy of mixed oligomers containing i-labeled monomers is inversely proportional to the number of labeled subunits per oligomer (Runnels and Scarlata limit). This method was very robust in describing the electrostatic interaction of Alexa Fluor 488 fluorescently labeled lysozyme (Lz-A488) with phosphatidylserine-containing membranes. The pronounced decrease detected in the fluorescence anisotropy of Lz-A488 always correlated with the system reaching a high membrane surface density of the protein (at a low lipid-to-protein (L/P) molar ratio). The occurrence of energy homo transfer-induced fluorescence depolarization was further confirmed by measuring the anisotropy decays of Lz-A488 under these conditions. A global analysis of the steady-state anisotropy data obtained under a wide range of experimental conditions (variable anionic lipid content of the liposomes, L/P molar ratios and protein fractional labeling) confirmed that membrane-bound Lz-A488 assembled into oligomeric complexes, possibly with a stoichiometry of k = 6 ± 1. This study illustrates that even in the presence of a coupled partition-oligomerization equilibrium, steady-state anisotropy measurements provide a simple and reliable tool to monitor the self-assembly of membrane-bound proteins.
影响因子：
2.9
作者：
E. Rusinova;V. Tretyachenko-Ladokhina;Oana Vele;D. F. Senear;J. B. Alexander Ross
通讯作者：
E. Rusinova;V. Tretyachenko-Ladokhina;Oana Vele;D. F. Senear;J. B. Alexander Ross
影响因子：
2.9
作者：
Robia, SL;Flohr, NC;Thomas, DD
通讯作者：
Thomas, DD