Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan.
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan; International Graduate Program of Molecular Science and Technology, National Taiwan University, Taipei, Taiwan.
Biophys J. 2022 May 17;121(10):1789-1798. doi: 10.1016/j.bpj.2022.04.022. Epub 2022 Apr 18.
Purple membrane (PM) is composed of several native lipids and the transmembrane protein bacteriorhodopsin (bR) in trimeric configuration. The delipidated PM (dPM) samples can be prepared by treating PM with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) to partially remove native lipids while maintaining bR in the trimeric configuration. By correlating the photocycle kinetics of bR and the exact lipid compositions of the various dPM samples, one can reveal the roles of native PM lipids. However, it is challenging to compare the lipid compositions of the various dPM samples quantitatively. Here, we utilize the absorbances of extracted retinal at 382 nm to normalize the concentrations of the remaining lipids in each dPM sample, which were then quantified by mass spectrometry, allowing us to compare the lipid compositions of different samples in a quantitative manner. The corresponding photocycle kinetics of bR were probed by transient difference absorption spectroscopy. We found that the removal rate of the polar lipids follows the order of BPG ≈ GlyC < S-TGD-1 ≈ PG < PGP-Me ≈ PGS. Since BPG and GlyC have more nonpolar phytanyl groups than other lipids at the hydrophobic tail, causing a higher affinity with the hydrophobic surface of bR, the corresponding removal rates are slowest. In addition, as the reaction period of PM and CHAPS increases, the residual amounts of PGS and PGP-Me significantly decrease, in concomitance with the decelerated rates of the recovery of ground state and the decay of intermediate M, and the reduced transient population of intermediate O. PGS and PGP-Me are the lipids with the highest correlation to the photocycle activity among the six polar lipids of PM. From a practical viewpoint, combining optical spectroscopy and mass spectrometry appears a promising approach to simultaneously track the functions and the concomitant active components in a given biological system.
紫膜由几种天然脂质和三聚体构象的跨膜蛋白细菌视紫红质（bR）组成。通过用 CHAPS（3-[(3-胆酰胺丙基)二甲氨基]-1-丙磺酸钠）处理紫膜来制备去垢剂处理的紫膜（dPM）样品，可以部分去除天然脂质，同时保持 bR 三聚体构象。通过关联 bR 的光循环动力学和各种 dPM 样品的确切脂质组成，可以揭示天然 PM 脂质的作用。然而，定量比较各种 dPM 样品的脂质组成具有挑战性。在这里，我们利用提取的视黄醛在 382nm 处的吸光度来归一化每个 dPM 样品中剩余脂质的浓度，然后通过质谱定量，从而可以定量比较不同样品的脂质组成。通过瞬态差示吸收光谱探测 bR 的相应光循环动力学。我们发现极性脂质的去除率遵循以下顺序：BPG≈GlyC<S-TGD-1≈PG<PGP-Me≈PGS。由于 BPG 和 GlyC 在疏水性尾部比其他脂质具有更多的非极性植烷基团，与 bR 的疏水性表面具有更高的亲和力，因此相应的去除率最慢。此外，随着 PM 和 CHAPS 的反应时间增加，PGS 和 PGP-Me 的残留量显著减少，同时中间 M 的恢复速度减慢，中间 O 的瞬态种群减少。PGS 和 PGP-Me 是 PM 六种极性脂质中与光循环活性相关性最高的脂质。从实际的角度来看，将光学光谱和质谱结合起来似乎是一种很有前途的方法，可以同时跟踪给定生物系统中的功能和伴随的活性成分。
