Extending molecular modeling methodology to study insertion of membrane nanopores.

C ompartmentalization is a key principle for the functional organization of living cells (1). Transport across compartmental boundaries and, in particular, across the cell wall is controlled by membrane proteins that act as selective channels and transporters. Puncturing the boundaries leads to pathologies, e.g., in the case of toxins, but is also an opportunity for treatment, e.g., in the case of antimicrobial peptides. The ability of membrane channels to sort single molecules is of great interest in bioengineering and, as a result, membrane channels like -hemolysin have been adopted for in vitro devices (2–5) or used as an inspiration for manufacturing artificial channels (6, 7). For deployment of proteins as channels, they need to be inserted into compartmental walls made of lipid bilayers. The majority of membrane proteins, socalled constitutive membrane proteins (8), are inserted into a lipid bilayer at the same time as they are synthesized. A special membrane protein, translocon (9), binds to the ribosome synthesizing the membrane protein and threads the protein into a lipid bilayer through an internal channel (10, 11). In contrast, the nonconstitutive membrane proteins, such as toxins (12) and antimicrobial peptides (13), insert themselves into a lipid bilayer without assistance. The insertion mechanism of such proteins, which is an indispensable part of their function, is poorly understood. The work of Lopez et al. (14) in this issue of PNAS demonstrates how a direct insertion of a typical nonconstitutive protein into a lipid membrane could take place. Modeling a membrane protein as a hydrophobic tube with hydrophilic sites at the tube’s ends, they observed, in an unprecedented molecular dynamics (MD) simulation, a spontaneous insertion of a generic nanosyringe into a lipid bilayer. The simulation revealed microscopic details of the insertion kinetics, showing the mechanism by which lipids assist one of the ends of the nanosyringe to cross the hydrophobic core of the membrane. The simulation monitored also the facility of the nanosyringe to conduct water molecules. The inserted nanosyringe is shown in Fig. 1. The time scale of the protein insertion process is beyond that covered by conventional microscopic simulations. Even when biased by an external electrical field, insertion of a small protein such as a single -helix could not be observed in all-atom MD simulations (15), because the lipid molecules forming the bilayer do not adjust their conformations quickly enough. Steered MD (16), which is a proven method to accelerate events along the path connecting the initial and final states of a process, cannot be deployed for studying a spontaneous insertion of a transmembrane