Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly ␣-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1 H and 13 C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal ␣-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism. Hepatitis C virus (HCV)8 infection is a major cause of human chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1). About 170 million individuals worldwide are chronically infected with HCV, and current therapy based on a combination of pegylated interferon and ribavirin is poorly tolerated and ineffective in 50% of patients. In this context, the ongoing search for new drugs and targets is very active, and the structural and functional characterization of the HCV viroporin p7 is essential for the molecular understanding of its role in HCV replication and for antiviral drug development.HCV is a highly variable enveloped positive-stranded RNA virus, and patient isolates are classified into seven genotypes and numerous subtypes (2, 3) within the genus Hepacivirus of the family Flaviviridae (4). The HCV genome encodes a polyprotein precursor,

We have investigated the thermodynamics of phospholipid flip-flop and desorption. Using a series of PC (phosphatidylcholine) lipids with different lengths of acyl tails, and number of unsaturated tails, we calculated potentials of mean force (PMFs) in atomistic molecular dynamics simulations. The PMFs describe the free energy for moving a single lipid molecule from water to the center of the respective lipid bilayer. The free energy to move the lipid from equilibrium to the bilayer center is assumed to be the free energy barrier for lipid flip-flop. We find that the free energy barrier for flip-flop is strongly dependent on the structure of the bilayer; ranging from 16 kJ mol À1 in the thin DLPC bilayer, to 90 kJ mol À1 in the DOPC bilayer. There are large deformations in the bilayers' structure, to accommodate the charged PC head group in the bilayer interior. We observe pore formation in all the bilayers, except for POPC and DOPC. The free energy for desorption is equal to the excess chemical potential of the lipid in the bilayer compared to bulk water. The increased chemical potential for PC lipids with longer acyl tails is in qualitative agreement with the critical micelle concentrations. We also determined PMFs for transferring water into the center of the series of lipid bilayers. Water has the same free energy of transfer to the center of all the bilayers, indicating the lipid PMFs differ due to bilayer deformations. Lipid bilayers are soft and deformable, allowing large structural changes, which are dependent on the composition of the bilayer. Our results show that similar PC lipids with only slightly different acyl tails, can have dramatically different thermodynamic behavior.