Chemical and Biomolecular Engineering

Membranes in the intracellular eubacterial parasite Chlamydia trachomatis consist of the elementary body (EB) and reticular body (RB), and contain methyl branches at the iso-and anteiso-positions for some phospholipid chains. Acyl chain branching is the focus of this study. Molecular dynamics simulations were used to study bilayers of 1-13-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (13-MpPPC), 1-14methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (14-MpPPC), and diphytanoylphosphatidylcholine (DPhPC). These three membranes were simulated at 323 K and simulations of DPhPC at 298 K were also performed for better comparison to existing experimental data. Two simulations of representative EB and RB membranes of C. trachomatis composed of nine different lipid components were performed at 310.15 K, to accurately reflect compositions determined by experiment and physiological conditions. Based on nearly 0.5 μs of simulation data, we report that branching increases average lipid surface area, area elastic moduli, and lipid axial relaxation times, while decreasing lipid chain order. Branching also has a distinct effect on electron density profiles. Due to their high cholesterol concentrations, the EB and RB membranes were found to have relatively high area elastic moduli, which may have important biological implications.

Molecular dynamics simulations and 31 P-NMR spin-lattice (R 1 ) relaxation rates from 0.022 to 21.1 T of fluid phase dipalmitoylphosphatidylcholine bilayers are compared. Agreement between experiment and direct prediction from simulation indicates that the dominant slow relaxation (correlation) times of the dipolar and chemical shift anisotropy spin-lattice relaxation are ;10 ns and 3 ns, respectively. Overall reorientation of the lipid body, consisting of the phosphorus, glycerol, and acyl chains, is well described within a rigid-body model. Wobble, with D ? ¼ 1-2 3 10 8 s À1 , is the primary component of the 10 ns relaxation; this timescale is consistent with the tumbling of a lipid-sized cylinder in a medium with the viscosity of liquid hexadecane. The value for D k ; the diffusion constant for rotation about the long axis of the lipid body, is difficult to determine precisely because of averaging by fast motions and wobble; it is tentatively estimated to be 1 3 10 7 s À1 . The resulting D k /D ? % 0.1 implies that axial rotation is strongly modulated by interactions at the lipid/water interface. Rigid-body modeling and potential of mean force evaluations show that the choline group is relatively uncoupled from the rest of the lipid. This is consistent with the ratio of chemical shift anisotropy and dipolar correlation times reported here and the previous observations that 31 P-NMR lineshapes are axially symmetric even in the gel phase of dipalmitoylphosphatidylcholine.

Lactose permease in E. coli (LacY) transports both anomeric states of disaccharides but has greater affinity for α-sugars. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions, binding structures, and global protein motions in response to sugar binding by investigating LacY (the experimental mutant and wild-type) embedded in a fully hydrated lipid bilayer. A total of twelve MD simulations of 20-25ns each with β(α)-D-galactopyranosyl-(1,1)-β-Dgalactopyranoside (ββ-(Galp) 2 ) and αβ-(Galp) 2 result in binding conformational families that depend on the anomeric state of the sugar. Both sugars strongly interact with Glu-126 and αβ-(Galp) 2 has a greater affinity to this residue. Binding conformations are also seen that involve protein residues not observed in the crystal structure, as well as those involved in the proton translocation (Phe-118, Asn-119, Asn-240, His-322, . Common to nearly all protein-sugar structures, water acts as a hydrogen bond bridge between the disaccharide and protein. The average binding energy is more attractive for αβ-(Galp) 2 than ββ-(Galp) 2 , i.e., −10.7±0.7 and −3.1±1.0 kcal/mol, respectively. Of the twelve helices in LacY, Helix-IV is the least stable with ββ-(Galp) 2 binding resulting in larger distortion than αβ-(Galp) 2 .

Based on the crystal structure of lactose permease (LacY) open to the cytoplasm, a hybrid molecular simulation approach with self-guided Langevin dynamics (SGLD) is used to describe conformational changes that lead to a periplasmic-open state. This hybrid approach consists of implicit (IM) and explicit (EX) membrane simulations and requires SGLD to enhance protein motions during the IM simulations. The pore radius of the lumen increases by 3.5 Å on the periplasmic side and decreases by 2.5 Å on the cytoplasmic side (relative to the crystal structure), which suggest a lumen that is fully open to the periplasm to allow for extracellular sugar transport and closed to the cytoplasm. Based on our simulations, the mechanism that triggers this conformational change to the periplasmic-open state is the protonation Glu269 and binding of the disaccharide. Then, helix packing is destabilized by breaking of several side chains involved in hydrogen bonding (Asn245, Ser41, Glu374, Lys42 and Gln242). For the periplasmic-open conformations obtained from our simulations, helix-helix distances agree well with experimental measurements using double electron-electron resonance, fluorescence resonance energy transfer, and varying sized cross-linkers. The periplasmic-open conformations are also in compliance with various substrate accessibility/reactivity measurements that indicate an opening of the protein lumen on the periplasmic side on sugar binding. The comparison with these measurements suggests a possible incomplete closure of the cytoplasmic half in our simulations. However, the closure is sufficient to prevent the disaccharide from transporting to the cytoplasm, which is in accordance with the well-established alternating access model. Ser53, Gln60, and Phe354 are determined to be important in sugar transport during the periplasmic-open stage of the sugar transport cycle and the sugar is found to undergo an orientational change in order to escape the protein lumen.

Sterols have been shown experimentally to bind to the Osh4 protein (a homolog of the oxysterol binding proteins) of Saccharomyces cerevisiae within a binding tunnel, which consists of antiparallel β-sheets that resemble a β-barrel and three α-helices of the N-terminus. This and other Osh proteins are essential for intracellular transport of sterols and ultimately cell life. Molecular dynamics (MD) simulations are used to study the binding of cholesterol to Osh4 at the atomic level. The structure of the protein is stable during the course of all MD simulations and has little deviation from the experimental crystal structure. The conformational stability of cholesterol within the binding tunnel is aided in part by direct or water-mediated interactions between the 3hydroxyl (3-OH) group of cholesterol and Trp 46 , Gln 96 , Tyr 97 , Asn 165 , and/or Gln 181 as well as dispersive interactions with Phe 42 , Leu 24 , Leu 39 , Ile 167 , and Ile 203 . These residues along with other nonpolar residues in the binding tunnel and lid contribute nearly 75% to the total binding energy. The strongest and most populated interaction is between Gln 96 and 3-OH with a cholesterol/Gln 96 interaction energy of □4.5±1.0 kcal/mol. Phe 42 has a similar level of attraction to cholesterol with □4.1±0.3 kcal/mol. A MD simulation without the N-terminus lid that covers the binding tunnel resulted in similar binding conformations and binding energies compared to simulations with the full-length protein. Steered MD was used to determine details of the mechanism used by Osh4 to release cholesterol to the cytoplasm. Phe 42 , Gln 96 , Asn 165 , Gln 181 , Pro 211 , and Ile 206 are found to direct the cholesterol as it exits the binding tunnel, as well as Lys 109 . The mechanism of sterol release is conceptualized as a molecular ladder with the rungs being amino acids or water-mediated amino acids that interact with 3-OH.

High-level ab initio quantum mechanical calculations are used to study various gauche conformational energies of n-pentane to n-decane. The destabilizing "pentane effect" (adjacent gauche states of opposite sign) for alkanes is confirmed, but the energies were found to depend slightly on chain length. In contrast, introducing an adjacent gauche of the same sign requires only 0.22-0.37 kcal/mol, approximately half of the single gauche state energy. This adjacent gauche stabilization should be taken into account when formulating or analyzing rotational isomeric models, carrying out conformational analysis, and developing force fields for alkanes, lipids, and related polymers.

The CHARMM-GUI Membrane Builder (http://www.charmm-gui.org/input/membrane), an intuitive, straightforward, web-based graphical user interface, was expanded to automate the building process of heterogeneous lipid bilayers, with or without a protein and with support for up to 32 different lipid types. The efficacy of these new features was tested by building and simulating lipid bilayers that resemble yeast membranes, composed of cholesterol, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine, palmitoyloleoylphosphatidylamine, and palmitoyloleoylphosphatidylserine. Four membranes with varying concentrations of cholesterol and phospholipids were simulated, for a total of 170 ns at 303.15 K. Unsaturated phospholipid chain concentration had the largest influence on membrane properties, such as average lipid surface area, density profiles, deuterium order parameters, and cholesterol tilt angle. Simulations with a high concentration of unsaturated chains (73%, membrane unsat) resulted in a significant increase in lipid surface area and a decrease in deuterium order parameters, compared with membranes with a high concentration of saturated chains (60-63%, membrane sat). The average tilt angle of cholesterol with respect to bilayer normal was largest, and the distribution was significantly broader for membrane unsat. Moreover, short-lived cholesterol orientations parallel to the membrane surface existed only for membrane unsat. The membrane sat simulations were in a liquid-ordered state, and agree with similar experimental cholesterol-containing membranes.

The fully hydrated liquid crystalline phase of the dimyristoylphosphatidycholine lipid bilayer at 30°C was simulated using molecular dynamics with the CHARMM potential for five surface areas per lipid (A) in the range 55-65 Å 2 that brackets the previously determined experimental area 60.6 Å 2. The results of these simulations are used to develop a new hybrid zero-baseline structural model, denoted H2, for the electron density profile, r(z), for the purpose of interpreting x-ray diffraction data. H2 and also the older hybrid baseline model were tested by fitting to partial information from the simulation and various constraints, both of which correspond to those available experimentally. The A, r(z), and F(q) obtained from the models agree with those calculated directly from simulation at each of the five areas, thereby validating this use of the models. The new H2 was then applied to experimental dimyristoylphosphatidycholine data; it yields A ¼ 60.6 6 0.5 Å 2 , in agreement with the earlier estimate obtained using the hybrid baseline model. The electron density profiles also compare well, despite considerable differences in the functional forms of the two models. Overall, the simulated r(z) at A ¼ 60.7 Å 2 agrees well with experiment, demonstrating the accuracy of the CHARMM lipid force field; small discrepancies indicate targets for improvements. Lastly, a simulation-based model-free approach for obtaining A is proposed. It is based on interpolating the area that minimizes the difference between the experimental F(q) and simulated F(q) evaluated for a range of surface areas. This approach is independent of structural models and could be used to determine structural properties of bilayers with different lipids, cholesterol, and peptides.

is an enzyme which controls spermidine/spermine concentrations, both of which are polyamines playing an important role for cell development. Recently we have investigated the effect of missense mutations (G56S, V132G and I150T) known to cause Synder-Robinson Syndrome (SRS) on SMS stability and dynamics. Here we extend our study to predict the mutability of these clinically observed sites. For this purpose, we substitute in silico the wild type residue at each site (G56, V132 and I150) with all other 19 amino acids and calculate the effect of stability and ionizations states of SMS. We show that mutations at site 150 are expected to greatly disrupt wide type function regardless of the amino acid substitution. Such site is termed intolerable site and is characterized with very low mutability. In contrast, site V132, despite being at the dimer interface is predicted to tolerate mutations and to be quite mutable. The G56 site is in the middle of the spectra, since some of the mutations are predicted to have significant effect on dimer stability, which in turn is crucial for the function of SMS. The performed analysis shows that mutability depends on the detail of the structural and functional factors and can not be predicted based on conservation of wild type properties alone. This work was supported by awards from NLM/NIH, grant numbers 1R03LM009748 and 1R03LM009748-S1. 1740-Pos Board B650 Properties of Lipid a Bilayers Analyzed by Molecular Dynamics Simulations Danielle Stuhlsatz, Richard Venable, Wonpil Im. Lipopolysaccharide (LPS), responsible for the toxicity of Gram-negative bacteria, is made up of three regions: the O antigen, the core, and the innermost lipid A. Lipid A is the lipid component whose hydrophobic nature allows LPS to anchor to the bacteria outer membrane. Utilizing the latest C36 CHARMM lipid and carbohydrate force field, we have constructed a lipid A molecule of E. coli. Various bilayer systems were then constructed six different conditions: 1) CaCl 2 at 80% water and at 303K or 323K, 2) KCl at 80% water at 303K or 323K, and 3) KCl at 40% water at 303K or 323K. We are currently running molecular dynamics simulations of these systems. The simulation results will be discussed in terms of how different temperatures, ratios of water molecules to lipids, and charged ions affects the lipid A bilayer properties such as surface area, ion location, chain order, bilayer thickness and shape, and the carbohydrate conformations. The results from these simulations will be also compared with available experimental data. This work will be the basis of the future simulations of the complex glycolipid simulations such as LPS, and membrane protein simulations in more native bilayer environments.

X-ray scattering is a promising tool with which to characterize systems of solidsupported membranes. There are many different scattering techniques used in the characterization, but all suffer from a necessarily low electron density contrast between the membrane and the water medium in which it must exist. Labeling membranes with a high-contrast scatterer such as gold is a promising avenue to solve this problem. In this work, silicon-supported membranes of 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were prepared by both standard Langmuir-Blodgett deposition and fusion of vesicles onto the substrate surface. Membranes are characterized using specular x-ray reflectometry, and modeled to fit physical systems. One percent by count 1,2-dipalmitoyl-sn-glycero-phosphoethanolamine (DPPE) with a gold tag attached was then added to both systems. Gold labeled membranes were then characterized and modeled. The effect of gold labeling is shown to characteristically change the membrane density profile in addition to enhancing density contrast between the membrane and the water medium.

Storage of large molecular dynamics (MD) simulation measurements in standard databases is a challenging task. The requirements on disk space, input/output (I/O) and data transfer bandwidth are excessively high due to the large volume, possibly terabytes or petabytes, of data generated. Storage of data in

Loop-gating is one of two voltage-dependent mechanisms that regulate the open probability of connexin channels. The loop-gate permeability barrier is formed by a segment of the first extracellular loop (E1) (the parahelix) and appears to be accompanied by straightening of the bend angle between E1 and the first transmembrane domain (TM1). Here, all-atom molecular dynamics simulations are used to identify and characterize interacting van der Waals and electrostatic networks that stabilize the parahelices and TM1/E1 bend angles of the open Cx26 hemichannel. Dynamic fluctuations in an electrostatic network in each subunit are directly linked to the stability of parahelix structure and TM1/E1 bend angle in adjacent subunits. The electrostatic network includes charged residues that are pore-lining and thus positioned to be voltage sensors. We propose that the transition to the closed state is initiated by voltage-driven disruption of the networks that stabilize the open-state parahelix configuration, allowing the parahelix to protrude into the channel pore to form the loop-gate barrier. Straightening of the TM1/E1 bend appears to be a consequence of the reorganization of the interacting networks that accompany the conformational change of the parahelix. The electrostatic network extends across subunit boundaries, suggesting a concerted gating mechanism.

Molecular dynamics (MD) simulations of dipalmitoylphosphatidylcholine (DPPC) bilayers composed of 72 and 288 lipids are used to examine system size dependence on dynamical properties associated with the particle-mesh Ewald (PME) treatment of electrostatic interactions. The lateral diffusion constant, D ℓ , is 2.92×10 −7 and 0.95×10 −7 cm 2 /s for 72 and 288 lipids, respectively. This dramatic finite size effect originates from the correlation length of lipid diffusion, which extends to next-nearest neighbors in the 288 lipid system. Consequently, diffusional events in smaller systems can propagate across the boundaries of the periodic box. The internal dynamics of lipids calculated from the PME simulations are independent of the system size. Specifically, reorientational correlation functions for the slowly relaxing phosphorous-glycerol hydrogen, phosphorous-nitrogen vectors, and more rapidly relaxing CH vectors in the aliphatic chains are equivalent for the 72 and 288 lipid simulations. A third MD simulation of a bilayer with 72 lipids using spherical force-shift electrostatic cutoffs resulted in interdigitated chains, thereby rendering this cutoff method inappropriate.

We also propose a website to host such a DBMS that can facilitate researchers to upload their data and perform analysis efficiently. Users can analyze their data using efficient functions implemented to access the data. Index structures are generated to store all results of analysis that may be interesting to other users, so that the results of analysis are readily available without the need to duplicate the analysis. The data upload feature can be made available through application program interfaces (APIs). Users can upload their data using the APIs. The DBMS takes care of generating indexes, storing results of analysis and retrieving efficiently, whenever users run analysis query or request information.