CNRS, Drug Resistance Mechanism and Modulation Laboratory, Ligue labeled team 2009, Lyon, France.
PLoS One. 2011 Mar 31;6(3):e18036. doi: 10.1371/journal.pone.0018036.
Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation.
METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein.
CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.
膜蛋白是药物研发的重要靶点，但基于结构的药物设计具有挑战性。一个主要原因是去污剂不能像天然脂质那样有效地稳定膜结构域，这通常会导致在蛋白质提取和纯化过程中部分甚至完全丧失活性/稳定性，并阻止蛋白质以活性构象结晶。
方法/主要发现：设计了阴离子杯[4]芳烃基去污剂（C4Cn，n=1-12），通过疏水相互作用和与膜蛋白细胞质-膜界面处的碱性残基形成的盐桥网络来稳定膜结构域。这些化合物表现为表面活性剂，形成 5-24nm 的胶束，临界胶束浓度（CMC）如预期的那样，对 pH 值在 0.05 至 1.5mM 之间的范围敏感。通过 1H NMR 滴定和表面张力滴定实验，证实了这些分子与碱性氨基酸的相互作用。它们从不同来源提取膜蛋白，行为温和，在某些情况下会导致部分提取。它们还保留蛋白质的功能，这一点在 BmrA（枯草芽孢杆菌多药耐药 ATP 蛋白）中得到了证明，BmrA 是一种膜多药转运 ATP 酶，对去污剂提取特别敏感。这些新的去污剂使 BmrA 能够以 12µM 的 Kd 结合柔红霉素，这一值与使用十二烷基麦芽糖苷（DDM）纯化后观察到的值相似。它们比 SDS（十二烷基硫酸钠）、FC12（Foscholine 12）或 DDM 更有效地保持 BmrA 的 ATP 酶活性（在药物外排后使蛋白重置为初始状态）。它们还能使 C4Cn 提取的蛋白在与 FC12 交换去污剂后保持功能状态。最后，它们促进膜蛋白的 3D 结晶。
结论/意义：这些化合物有望以功能状态提取符合正内规则的膜蛋白。在这种情况下，它们可能有助于膜蛋白结晶领域。
