单分子荧光共振能量转移监测P-糖蛋白的药物转运机制
Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer
摘要：
在这项工作中，我们监测的P-糖蛋白（Pgp）的催化机制，使用单分子荧光共振能量转移（FRET）。Pgp是转运蛋白的ATP结合盒家族的成员，存在于动物细胞的质膜中，在那里它参与ATP水解驱动的疏水分子的输出。当表达于癌细胞的质膜时，Pgp的转运活性可通过将大多数疏水性药物从细胞内部排除而导致化疗失败。尽管正在进行的努力，Pgp耦合MgATP的结合和水解的药物分子的跨脂质双层易位的催化机制知之甚少。利用定点突变技术，我们将半胱氨酸残基引入Pgp的核苷酸结合域（NBD）的不同区域进行荧光标记。双标记的单个Pgp分子在药物刺激的ATP水解过程中表现出波动的FRET效率，这表明NBD在催化过程中发生了显着的运动。应用占空比优化的交替激光激发（DCO-ALEX）以最小化FRET伪影并选择适当的分子。数据表明，Pgp是一种高度动态的酶，在稳态周转期间似乎在至少两种主要构象之间波动。
英文摘要：
In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.
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