Membrane Interactions of Cy3 and Cy5 Fluorophores and Their Effects on Membrane-Protein Dynamics

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.