A straightforward method that accounts for the long-range Lennard-Jones (LJ) terms in constant pressure molecular dynamics simulations is presented. This long-range correction (LRC) consists of an additional applied pressure tensor which is periodically calculated from the difference of instantaneous pressures at the selected cutoff and a very long cutoff. It provides results that are nearly independent of the LJ cutoff distance at negligible additional calculation costs, and is particularly suited for anisotropic systems such as liquid/ liquid interfaces or heterogeneous macromolecules where approximations based on spherically symmetric radial distribution functions are expected to fail. The utility of the method is demonstrated for a series of alkanes and water, and for interfaces including a lipid bilayer. The LRC increases densities and decreases isothermal compressibilities, with the changes larger for alkanes than for water (where the long-range interactions are dominated by electrostatic interactions). While implementation of the LRC will not necessarily improve agreement with a particular experiment, it will provide a baseline for improvements in a parameter set that are consistent with the long-range Lennard-Jones interactions.

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The present study is an application of an approach recently developed by the authors for describing the structure of the hydrocarbon chains of lipid-bilayer membranes (LBMs) around embedded protein inclusions ( Biophys. J. 79:2867-2879). The approach is based on statistical mechanical integral equation theories developed for the study of dense liquids. First, the configurations extracted from molecular dynamics simulations of pure LBMs are used to extract the lateral density-density response function. Different pure LBMs composed of different lipid molecules were considered: dioleoyl phosphatidylcholine (DOPC), palmitoyl-oleoyl phosphatidylcholine (POPC), dipalmitoyl phosphatidylcholine (DPPC), and dimyristoyl phosphatidylcholine (DMPC). The results for the lateral density-density response function was then used as input in the integral equation theory. Numerical calculations were performed for protein inclusions of three different sizes. For the sake of simplicity, protein inclusions are represented as hard smooth cylinders excluding the lipid hydrocarbon core from a small cylinder of 2.5 A radius, corresponding roughly to one aliphatic chain, a medium cylinder of 5 A radius, corresponding to one alpha-helix, and a larger cylinder of 9 A radius, representing a small protein such as the gramicidin channel. The lipid-mediated interaction between protein inclusions was calculated using a closed-form expression for the configuration-dependent free energy. This interaction was found to be repulsive at intermediate range and attractive at short range for two small cylinders in POPC, DPPC, and DMPC bilayers, whereas it oscillates between attractive and repulsive values in DOPC bilayers. For medium size cylinders, it is again repulsive at intermediate range and attractive at short range, but for every model LBM considered here. In the case of a large cylinder, the lipid-mediated interaction was shown to be repulsive for both short and long ranges for the DOPC, POPC, and DPPC bilayers, whereas it is again repulsive and attractive for DMPC bilayers. The results indicate that the packing of the hydrocarbon chains around protein inclusions in LBMs gives rise to a generic (i.e., nonspecific) lipid-mediated interaction which favors the association of two alpha-helices and depends on the lipid composition of the membrane.

The structure of oxygenated trHbN from Mycobacterium tuberculosis shows an extended heme distal hydrogen-bond network that includes Tyr33(B10), Gln58(E11), and the bound O(2). In addition, trHbN structure shows a network of hydrophobic cavities organized in two orthogonal branches. In the present work, the structure and the dynamics of oxygenated and deoxygenated trHbN in explicit water was investigated from 100 ns molecular dynamics (MD) simulations. Results show that, depending on the presence or the absence of a coordinated O(2), the Tyr33(B10) and Gln58(E11) side chains adopt two different configurations in concert with hydrogen bond network rearrangement. In addition, our data indicate that Tyr33(B10) and Gln58(E11) control the dynamics of Phe62(E15). In deoxy-trHbN, Phe62(E15) is restricted to one conformation. Upon O(2) binding, the conformation of Gln58(E11) changes and residue Phe62(E15) fluctuates between two conformations. We also conducted a systematic study of trHbN tunnels by analyzing thousands of MD snapshots with CAVER. The results show that tunnel formation is the result of the dynamic reshaping of short-lived hydrophobic cavities. The analyses indicate that the presence of these cavities is likely linked to the rigid structure of trHbN and also reveal two tunnels, EH and BE, that link the protein surface to the buried distal heme pocket and not present in the crystallographic structure. The cavities are sufficiently large to accomodate and store ligands. Tunnel dynamics in trHbN was found to be controlled by the side-chain conformation of the Tyr33(B10), Gln58(E11), and Phe62(E15) residues. Importantly, in contrast to recently published works, our extensive systematic studies show that the presence or absence of a coordinated dioxygen does not control the opening of the long tunnel but rather the opening of the EH tunnel. In addition, the data lead to new and distinctly different conclusion on the impact of the Phe62(E15) residue on trHbN tunnels. We propose that the EH and the long tunnels are used for apolar ligands storage. The trajectories bring important new structural insights related to trHbN function and to ligand diffusion in proteins.

Duchenne muscular dystrophy (DMD), a severe hereditary disease affecting 1 in 3,500 boys, mainly results from the deletion of exon(s), leading to a reading frameshift of the DMD gene that abrogates dystrophin protein synthesis. Pairs of sgRNAs for the Cas9 of Staphylococcus aureus were meticulously chosen to restore a normal reading frame and also produce a dystrophin protein with normally phased spectrin-like repeats (SLRs), which is not usually obtained by skipping or by deletion of complete exons. This can, however, be obtained in rare instances where the exon and intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion) are at similar positions in the SLR. We used pairs of sgRNAs targeting exons 47 and 58, and a normal reading frame was restored in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. Restoration of the DMD reading frame and restoration of dystrophin expression were also obtained in vivo in the heart of the del52hDMD/mdx. Our results provide a proof of principle that SaCas9 could be used to edit the human DMD gene and could be considered for further development of a therapy for DMD.