Organic fluorophores, such as Cy3 and Cy5, have been widely used as chemical labels to probe the structure and dynamics of membrane proteins. Although a number of previous studies have reported on the possibility of some of the water-soluble fluorophores to interact with lipid bilayers, detailed fluorophore-lipid interactions and, more importantly, the potential effect of such interactions on the natural dynamics of the labeled membrane proteins have not been well studied. We have performed a large set of all-atom molecular dynamics simulations employing the highly mobile membrane mimetic model to describe spontaneous partitioning of the fluorophores into lipid bilayers with different lipid compositions. Spontaneous membrane partitioning of Cy3 and Cy5 fluorophores captured in these simulations proceeds in two steps. Electrostatic interaction between the fluorophores and the lipid headgroups facilitates the initial, fast membrane association of the fluorophores, followed by slow insertion of hydrophobic moieties into the lipid bilayer core. After the conversion of the resulting membrane-bound systems to full-membrane representations, biased-exchange umbrella sampling simulations are performed for free energy calculations, revealing a higher energy barrier for partitioning into negatively charged (phosphatidylserine or phosphatidylcholine) membranes than purely zwitterionic (phosphatidylcholine or phosphatidylethanolamine) ones. Furthermore, the potential effect of fluorophore-lipid interactions on membrane proteins has been examined by covalently linking Cy5 to single- and multipass transmembrane helical proteins. Equilibrium simulations show strong position-dependent effects of Cy5-tagging on the structure and natural dynamics of membrane proteins. Interactions between the tagged protein and Cy5 were also observed. Our results suggest that fluorophore-lipid interactions can affect the structure and dynamics of membrane proteins to various extents, especially in systems with higher structural flexibility.

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Cy3/Cy5 荧光团的膜相互作用及其对膜蛋白动力学的影响

Cy3 和 Cy5 等有机荧光团已被广泛用作化学标记来探测膜蛋白的结构和动力学。虽然之前的一些研究报告了一些水溶性荧光团与脂质双层相互作用的可能性，详细的荧光团-脂质相互作用，更重要的是，这种相互作用对标记膜蛋白的自然动力学的潜在影响没有得到很好的研究。我们已经进行了大量的全原子分子动力学模拟，使用高度移动的膜模拟模型来描述荧光团自发分配到具有不同脂质成分的脂质双层中。在这些模拟中捕获的 Cy3 和 Cy5 荧光团的自发膜分配分两步进行。荧光团和脂质头基之间的静电相互作用促进了荧光团的初始快速膜结合，随后疏水部分缓慢插入脂质双层核心。在将所得膜结合系统转换为全膜表示后，进行了自由能计算的偏置交换伞采样模拟，揭示了分配成带负电（磷脂酰丝氨酸或磷脂酰胆碱）膜的能量屏障高于纯两性离子（磷脂酰胆碱）膜或磷脂酰乙醇胺）。此外，通过将 Cy5 与单程和多程跨膜螺旋蛋白共价连接，研究了荧光团-脂质相互作用对膜蛋白的潜在影响。平衡模拟显示 Cy5 标记对膜蛋白的结构和自然动力学具有很强的位置依赖性影响。还观察到标记蛋白和 Cy5 之间的相互作用。我们的结果表明，荧光团-脂质相互作用可以在不同程度上影响膜蛋白的结构和动力学，特别是在具有更高结构灵活性的系统中。