Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, and Zernike Institute for Advanced Materials, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Structure. 2011 Mar 9;19(3):430-9. doi: 10.1016/j.str.2010.12.016.
Dual-color fluorescence-burst analysis (DCFBA) was applied to measure the quaternary structure and high-affinity binding of the bacterial motor protein SecA to the protein-conducting channel SecYEG reconstituted into lipid vesicles. DCFBA is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. SecA binds to SecYEG as a dimer with a nucleotide- and preprotein-dependent dissociation constant. One of the SecA protomers binds SecYEG in a salt-resistant manner, whereas binding of the second protomer is salt sensitive. Because protein translocation is salt sensitive, we conclude that the dimeric state of SecA is required for protein translocation. A structural model for the dimeric assembly of SecA while bound to SecYEG is proposed based on the crystal structures of the Thermotoga maritima SecA-SecYEG and the Escherichia coli SecA dimer.
双色荧光爆发分析（DCFBA）被应用于测量细菌运动蛋白 SecA 与再组装到脂质体中的蛋白质导通道 SecYEG 的四级结构和高亲和力结合。DCFBA 是一种平衡技术，可在单分子水平上直接观察和量化蛋白质-蛋白质相互作用。SecA 以依赖核苷酸和前体蛋白的解离常数结合 SecYEG 作为二聚体。SecA 的一个原体以耐盐的方式结合 SecYEG，而第二个原体的结合则对盐敏感。由于蛋白质易位对盐敏感，我们得出结论，SecA 的二聚体状态是蛋白质易位所必需的。基于 Thermotoga maritima SecA-SecYEG 和 Escherichia coli SecA 二聚体的晶体结构，提出了 SecA 与 SecYEG 结合时二聚体组装的结构模型。
