SUMMARY: A fundamental feature of cellular plasma membranes (PM) is asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM leaflets, nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being ~2-fold more unsaturated than the exoplasmic. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in asymmetric structures of protein transmembrane domains (TMD). These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.

Functioning as key players in cellular regulation of membrane curvature, BAR-domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface, and are held together by promiscuous interactions between endophilin's amphipathic N-terminal helices. Coarse-grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic, and that the N-terminal helices are required for formation of a stable and regular scaffold. Furthermore, endophilin accommodates different curvatures through a quantized addition or removal of endophilin dimers, which in some cases causes dimerization of endophilin's SH3 domains, suggesting that the spatial presentation of SH3-domains rather than affinity governs the recruitment of downstream interaction partners.

Molecular dynamics simulations reveal substructures within the liquid-ordered phase of lipid bilayers. These substructures, identified in a 10 μsec all-atom trajectory of liquid-ordered/liquid-disordered coexistence (Lo/Ld), are composed of saturated hydrocarbon chains packed with local hexagonal order, and separated by interstitial regions enriched in cholesterol and unsaturated chains. Lipid hydrocarbon chain order parameters calculated from the Lo phase are in excellent agreement with 2H NMR measurements; the local hexagonal packing is also consistent with 1H-MAS NMR spectra of the Lo phase, NMR diffusion experiments, and small angle X-ray- and neutron scattering. The balance of cholesterol-rich to local hexagonal order is proposed to control the partitioning of membrane components into the Lo regions. The latter have been frequently associated with formation of so-called rafts, platforms in the plasma membranes of cells that facilitate interaction between components of signaling pathways.

There are several examples of membrane-associated protein domains that target curved membranes. This behavior is believed to have functional significance in a number of essential pathways, such as clathrin-mediated endocytosis, which involve dramatic membrane remodeling and require the recruitment of various cofactors at different stages of the process. This work is motivated in part by recent experiments that demonstrated that the amphipathic N-terminal helix of endophilin (H0) targets curved membranes by binding to hydrophobic lipid bilayer packing defects which increase in number with increasing membrane curvature. Here we use state-of-the-art atomistic simulation to explore the packing defect structure of curved membranes, and the effect of this structure on the folding of H0. We find that not only are packing defects increased in number with increasing membrane curvature, but also that their size distribution depends nontrivially on the curvature, falling off exponentially with a decay constant that depends on the curvature, and crucially that even on highly curved membranes defects large enough to accommodate the hydrophobic face of H0 are never observed. We furthermore find that a percolation model for the defects explains the defect size distribution, which implies that larger defects are formed by coalescence of noninteracting smaller defects. We also use the recently developed metadynamics algorithm to study in detail the effect of such defects on H0 folding. It is found that the comparatively larger defects found on a convex membrane promote H0 folding by several kcal/mol, while the smaller defects found on flat and concave membrane surfaces inhibit folding by kinetically trapping the peptide. Together, these observations suggest H0 folding is a cooperative process in which the folding peptide changes the defect structure relative to an unperturbed membrane.

We extend replica exchange simulation in two ways, and apply our approaches to biomolecules. The first generalization permits exchange simulation between models of differing resolutioni.e., between detailed and coarse-grained models. Such "resolution exchange" can be applied to molecular systems or spin systems. The second extension is to "pseudo-exchange" simulations, which require little CPU usage for most levels of the exchange ladder and also substantially reduces the need for overlap between levels. Pseudo exchanges can be used in either replica or resolution exchange simulations. We perform efficient, converged simulations of a 50-atom peptide to illustrate the new approaches.Accepted for publication in physical review letters. PACS numbers:The simulation of biomolecules with 10 4 − 10 5 degrees of freedom has become routine, thanks to the accessibility of powerful computing resources, the development of reliable simulation software, and standardized empirical potential energy functions. For many biological applications, such as binding free energy estimation, it is desirable to generate an equilibrated ensemble of conformations. In principle, standard Monte Carlo (MC) and molecular dynamics (MD) algorithms are perfectly ergodic, and therefore will eventually generate such ensembles. In practice, the µ sec − sec timescale, which describes biologically relevant fluctuations, is not within reach of computation even for small proteins.Two broad strategies have been developed to address this problem. In one approach, dating to the earliest computational studies of proteins[1, 2], coarse-grained protein representations are adopted. This strategy continues to be popular [3,4].A second class of strategies attempts directly to enhance sampling of atomic-resolution models, including multiple time step methods [5,6], replica exchange [7]/parallel tempering [8,9,10], and other generalized ensemble techniques [11]. Parallel tempering (PT), which employs a ladder of replicas simulated at increasing temperatures, is widely used for state-of-the-art molecular dynamics simulations, but presently is limited to small proteins [12], as the resources required increase rapidly with the system size.This Letter presents two new tools for biomolecular simulation, by extending the PT approach and exploiting the speed of coarse-grained models. The first extension is a "resolution exchange" (res-ex) algorithm whichinstead of using high-temperature simulation to increase sampling, as does PT -uses inexpensive coarse-grained * elyman@ccbb.pitt.edu † dmz@ccbb.pitt.edu models to cross barriers. Boltzmann-weighted ensembles are produced. The algorithm is implemented in close analogy to PT, and can also be applied to magnetic systems (e.g., the Ising model). The res-ex approach is natural for proteins, and indeed the kernel of the idea was suggested in the early days of protein simulation [1]. More recently, the approach has been implemented in an ad hoc way, without proper statistical weighting [3]. A rigorous method to calculate free en...

All-atom simulation data are presented for ternary mixtures of palmitoyl sphingomyelin (PSM), cholesterol, and either palmitoyl oleoyl phosphatidyl choline or dioleoyl phosphatidyl choline (DOPC). For comparison, data for a mixture of dipalmitoyl phosphatidyl choline (DPPC), cholesterol, and DOPC are also presented. Compositions corresponding to the liquid-ordered phase, the liquid-disordered phase, and coexistence of the two phases are simulated for each mixture. Within the liquid-ordered phase, cholesterol is preferentially solvated by DOPC if it is available, but if DOPC is replaced by POPC, cholesterol is preferentially solvated by PSM. In the DPPC mixtures, cholesterol interacts preferentially with the saturated chains via its smooth face, whereas in the PSM mixtures, cholesterol interacts preferentially with PSM via its rough face. Interactions between cholesterol and PSM have a very particular character: hydrogen bonding between cholesterol and the amide of PSM rotates the tilt of the amide plane, which primes it for more robust hydrogen bonding with other PSM. Cholesterol-PSM hydrogen bonding also locally modifies the hexagonal packing of hydrocarbon chains in the liquid-ordered phase of PSM mixtures.

We present a method to parameterize heterogeneous elastic network models (heteroENMs) of proteins to reproduce the fluctuations observed in atomistic simulations. Because it is based on atomistic simulation, our method allows the development of elastic coarse-grained models of proteins under different conditions or in different environments. The method is simple and applicable to models at any level of coarse-graining. We validated the method in three systems. First, we computed the persistence length of ADP-bound F-actin, using a heteroENM model. The value of 6.1 +/- 1.6 microm is consistent with the experimentally measured value of 9.0 +/- 0.5 microm. We then compared our method to a uniform elastic network model and a realistic extension algorithm via covariance Hessian (REACH) model of carboxy myoglobin, and found that the heteroENM method more accurately predicted mean-square fluctuations of alpha-carbon atoms. Finally, we showed that the method captures critical differences in effective harmonic interactions for coarse-grained models of the N-terminal Bin/amphiphysin/Rvs (N-BAR) domain of amphiphysin, by building models of N-BAR both bound to a membrane and free in solution.

Assessing the convergence of a biomolecular simulation is an essential part of any careful computational investigation, because many fundamental aspects of molecular behavior depend on the relative populations of different conformers. Here we present a physically intuitive method to self-consistently assess the convergence of trajectories generated by molecular dynamics and related methods. Our approach reports directly and systematically on the structural diversity of a simulation trajectory. Straightforward clustering and classification steps are the key ingredients, allowing the approach to be trivially applied to systems of any size. Our initial study on met-enkephalin strongly suggests that even fairly long trajectories (approximately 50 ns) may not be converged for this small--but highly flexible--system.