No abstract

We have reparameterized the dihedral parameters in a commonly used united-atom lipid force field so that they can be used with the all-atom OPLS force field for proteins implemented in the molecular dynamics simulation software GROMACS. Simulations with this new combination give stable trajectories and sensible behaviour of both lipids and protein. We have calculated the free energy of transfer of amino acid side chains between water and 'lipid-cyclohexane', made of lipid force field methylene groups, as a hydrophobic mimic of the membrane interior, for both the OPLS-AA and a modified OPLS-AA force field which gives better hydration free energies under simulation conditions close to those preferred for the lipid force field. The average error is 4.3 kJ mol(-1) for water-'lipid-cyclohexane' compared to 3.2 kJ mol(-1) for OPLS-AA cyclohexane and 2.4 kJ mol(-1) for the modified OPLS-AA water-'lipid-cyclohexane'. We have also investigated the effect of different methods to combine parameters between the united-atom lipid force field and the united-atom protein force field ffgmx. In a widely used combination, the strength of interactions between hydrocarbon lipid tails and proteins is significantly overestimated, causing a decrease in the area per lipid and an increase in lipid ordering. Using straight combination rules improves the results. Combined, we suggest that using OPLS-AA together with the united-atom lipid force field implemented in GROMACS is a reasonable approach to membrane protein simulations. We also suggest that using partial volume information and free energies of transfer may help to improve the parameterization of lipid-protein interactions and point out the need for accurate experimental data to validate and improve force field descriptions of such interactions.

Protonated networks of internal water molecules appear to be involved in proton transfer in various integral membrane proteins. High-resolution x-ray studies of protein crystals at low temperature deliver mean positions of most internal waters, but only limited information about fluctuations within such H-bonded networks formed by water and residues. The question arises as to how water molecules behave inside and on the surface of a fluctuating membrane protein under more physiological conditions. Therefore, as an example, long-time molecular dynamics simulations of bacteriorhodopsin were performed with explicit membrane/water environment. Based on a recent x-ray model the bacteriorhodopsin trimer was inserted in a fully solvated 16 x 16 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-bilayer patch, resulting in a system of approximately 84,000 atoms. Unrestrained molecular dynamics calculations of 5 ns were performed using the GROMACS package and force field. Mean water densities were computed to describe the anisotropic distribution of internal water molecules. In the whole protein two larger areas of higher water density are identified. They are located between the central proton binding site, the Schiff base, and the extracellular proton release site. Separated by Arg-82 these water clusters could provide a proton release pathway in a Grotthus-like mechanism as indicated by a continuum absorbance change observed during the photocycle by time-resolved Fourier transform infrared spectroscopy. Residues are identified which are H-bonded to the water clusters and are therefore facilitating proton conduction. Their influence on proton transfer via the H-bonded network as indicated by the continuum absorbance change is predicted. This may explain why several site-directed mutations alter the proton release kinetics without a direct involvement in proton transfer.

ATP-binding cassette (ABC) transporters are integral membrane proteins that actively transport molecules across cell membranes. In Escherichia coli they consist primarily of import systems that involve in addition to the ABC transporter itself a substrate binding protein and outer membrane receptors or porins, and a number of transporters with varied functions. Recent crystal structures of a number of ATPase domains, substrate binding proteins, and full-length transporters have given new insight in the molecular basis of transport. Bioinformatics approaches allow an approximate identification of all ABC transporters in E. coli and their relation to other known transporters. Computational approaches involving modeling and simulation are beginning to yield insight into the dynamics of the transporters. We summarize the function of the known ABC transporters in E. coli and mechanistic insights from structural and computational studies.

Playing a major role in the expulsion of antibiotics and the secretion of cell toxins in conjunction with inner membrane transporters of three protein superfamilies, the outer membrane channel TolC occurs in at least two states blocking or permitting the passage of substrates. The details of the underlying gating mechanism are not fully understood. Addressing the questions of extracellular access control and periplasmic gating mechanism, we conducted a series of independent, unbiased 150-300 ns molecular dynamics simulations of wild-type TolC in a phospholipid membrane/150 mM NaCl water environment. We find that TolC opens and closes freely on the extracellular side, suggesting the absence of a gating mechanism on this side in the isolated protein. On the periplasmic side, we observe the outer periplasmic bottleneck region adopting in all simulations a conformation more open than the TolC wild-type crystal structures until in one run the successive binding of two sodium ions induces the transition to a conformation more closed than any of the available TolC X-ray structures. Concurrent with a heightened sodium residence probability near Asp374, the inner periplasmic bottleneck region at Asp374 remains closed throughout the simulations unless all NaCl is removed from the system, inducing a reopening of the outer and inner bottleneck. Our findings suggest that TolC is locked only on the periplasmic side in a sodium-dependent manner.