Nonadditive Compositional Curvature Energetics of Lipid Bilayers.

The unique properties of the individual lipids that compose biological membranes together determine the energetics of the surface. The energetics of the surface, in turn, govern the formation of membrane structures and membrane reshaping processes, and thus they will underlie cellular-scale models of viral fusion, vesicle-dependent transport, and lateral organization relevant to signaling. The spontaneous curvature, to the best of our knowledge, is always assumed to be additive. We describe observations from simulations of unexpected nonadditive compositional curvature energetics of two lipids essential to the plasma membrane: sphingomyelin and cholesterol. A model is developed that connects molecular interactions to curvature stress, and which explains the role of local composition. Cholesterol is shown to lower the number of effective Kuhn segments of saturated acyl chains, reducing lateral pressure below the neutral surface of bending and favoring positive curvature. The effect is not observed for unsaturated (flexible) acyl chains. Likewise, hydrogen bonding between sphingomyelin lipids leads to positive curvature, but only at sufficient concentration, below which the lipid prefers negative curvature.

CHARMM: the biomolecular simulation program.

CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecular simulation program. It has been developed over the last three decades with a primary focus on molecules of biological interest, including proteins, peptides, lipids, nucleic acids, carbohydrates, and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estimators, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. The CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numerous platforms in both serial and parallel architectures. This article provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM article in 1983.

Molecular dynamics simulations of a lipid bilayer and of hexadecane: an investigation of membrane fluidity.

Molecular dynamics simulations of a fluid-phase dipalmitoyl phosphatidylcholine lipid bilayer in water and of neat hexadecane are reported and compared with nuclear magnetic resonance spin-lattice relaxation and quasi-elastic neutron scattering data. On the 100-picosecond time scale of the present simulations, there is effectively no difference in the reorientational dynamics of the carbons in the membrane interior and in pure hexadecane. Given that the calculated fast reorientational correlation times and the "microscopic" lateral diffusion of the lipids show excellent agreement with the experimental results, it is concluded that the apparently high viscosity of the membrane is more closely related to molecular interactions on the surface rather than in the interior.

Solution structure and dynamics of linked cell attachment modules of mouse fibronectin containing the RGD and synergy regions: comparison with the human fibronectin crystal structure.

We report the three-dimensional solution structure of the mouse fibronectin cell attachment domain consisting of the linked ninth and tenth type III modules, mFnFn3(9,10). Because the tenth module contains the RGD cell attachment sequence while the ninth contains the synergy region, mFnFn3(9,10) has the cell attachment activity of intact fibronectin. Essentially complete signal assignments and approximately 1800 distance and angle restraints were derived from multidimensional heteronuclear NMR spectra. These restraints were used with a hybrid distance geometry/simulated annealing protocol to generate an ensemble of 20 NMR structures having no distance or angle violations greater than 0.3 A or 3 degrees. Although the beta-sheet core domains of the individual modules are well-ordered structures, having backbone atom rmsd values from the mean structure of 0.51(+/-0.12) and 0.40(+/-0.07) A, respectively, the rmsd of the core atom coordinates increases to 3.63(+/-1.41) A when the core domains of both modules are used to align the coordinates. The latter result is a consequence of the fact that the relative orientation of the two modules is not highly constrained by the NMR restraints. Hence, while structures of the beta-sheet core domains of the NMR structures are very similar to the core domains of the crystal structure of hFnFn3(9,10), the ensemble of NMR structures suggests that the two modules form a less extended and more flexible structure than the fully extended rod-like crystal structure. The radius of gyration, Rg, of mFnFn3(9,10) derived from small-angle neutron scattering measurements, 20.5(+/-0.5) A, agrees with the average Rg calculated for the NMR structures, 20.4 A, and is ca 1 A less than the value of Rg calculated for the X-ray structure. The values of the rotational anisotropy, D ||/D perpendicular, derived from an analysis of 15N relaxation data, range from 1.7 to 2.1, and are significantly less than the anisotropy of 2.67 predicted by hydrodynamic modeling of the crystal coordinates. In contrast, hydrodynamic modeling of the NMR coordinates yields anisotropies in the range of 1.9 to 2.7 (average 2.4(+/-0.2)), with NMR structures bent by more than 20 degrees relative the crystal structure having calculated anisotropies in best agreement with experiment. In addition, the relaxation parameters indicate that several loops in mFnFn3(9,10), including the RGD loop, are flexible on the nanosecond to picosecond time-scale. Taken together, our results suggest that, in solution, the limited set of interactions between the mFnFn3(9,10) modules position the RGD and synergy regions to interact specifically with cell surface integrins, and at the same time permit sufficient flexibility that allows mFnFn3(9,10) to adjust for some variation in integrin structure or environment.

Theoretically determined three-dimensional structures for amphipathic segments of the HIV-1 gp41 envelope protein.

Three-dimensional computer models for two segments of the C terminus of gp41, the transmembrane AIDS envelope protein, which may form amphipathic alpha-helices, have been generated using structure prediction techniques combined with energy minimization and molecular dynamics simulations. Regions gp41(772-790) and gp41(828-848) of the HXB2 strain of HIV-1 display extraordinarily high hydrophobic moment maxima as alpha-helices and when in an antiparallel conformation exhibit charge complementarity, implying that they may bind with each other and associate with the membrane. The feasibility of this hypothesis was tested in a series of computer simulations of these peptides, extended by several residues to include additional charge pairing. Beginning with a trial structure in the form of antiparallel alpha-helices of segments 770-794 and 824-856, systematic axial rotations and displacements were used to generate alternative initial states. Molecular dynamics simulations with alpha-helical torsional restraints yielded several approximately cylindrical dimeric structures highly stabilized by numerous salt links and other hydrogen bonds. This suggests that these two regions may fold back on each other in antiparallel fashion to form a loop in the tertiary structure over residues 770-856, with the loop closed by membrane-associated amphipathic alpha-helices with charged sides facing each other. We speculate that such structures could aggregate to form channels or otherwise destabilize the membrane, thereby contributing to the cytopathic effects of the gp120-gp41 complex.

Separation and quantitation of insulins and related substances in bulk insulin crystals and in injectables by reversed-phase high performance liquid chromatography and the effect of temperature on the separation.

The Food and Drug Administration performs potency assays of insulin products as part of the insulin certification program required by the Code of Federal Regulations. The official method specified in the United States Pharmacopeia (USP) is a bioassay measuring the depression of blood sugar concentrations in rabbits treated with the insulin under test and the official standard. The insulin products resulting from today's technology include highly purified isolates from bovine or porcine pancreas and insulin that is identical in structure to human insulin. A study of the effects of temperature on the separation of the components in insulin injectables led to the development of a reversed-phase high performance liquid chromatographic (HPLC) method that uses a sulfate buffer/acetonitrile mobile phase at 40 degrees C for the separation and quantitation of bovine, porcine, and human insulins and related substances. This HPLC method reduces analysis time to 1/60 of that required for the bioassay and yields more information about purity than the percent nitrogen determination that is one of the USP official procedures. The results of HPLC analyses were compared with those for the bioassay by means of a potency/area conversion factor computed on a species by species basis. Results for the bioassays and the HPLC determinations for 40 lots of bulk crystalline insulin were compared in this study; in general, the HPLC estimates fell within the 95% confidence interval for combined independent bioassays.

Molecular dynamics studies of the conformation of sorbitol.

Molecular dynamics simulations of a 3 molal aqueous solution of D-sorbitol (also called D-glucitol) have been performed at 300 K, as well as at two elevated temperatures to promote conformational transitions. In principle, sorbitol is more flexible than glucose since it does not contain a constraining ring. However, a conformational analysis revealed that the sorbitol chain remains extended in solution, in contrast to the bent conformation found experimentally in the crystalline form. While there are 243 staggered conformations of the backbone possible for this open-chain polyol, only a very limited number were found to be stable in the simulations. Although many conformers were briefly sampled, only eight were significantly populated in the simulation. The carbon backbones of all but two of these eight conformers were completely extended, unlike the bent crystal conformation. These extended conformers were stabilized by a quite persistent intramolecular hydrogen bond between the hydroxyl groups of carbon C-2 and C-4. The conformational populations were found to be in good agreement with the limited available NMR data except for the C-2-C-3 torsion (spanned by the O-2-O-4 hydrogen bond), where the NMR data support a more bent structure.

Data transmission through the telephone network: protocols, pitfalls, and some examples.

Invariably, the situation arises where it is desirable to transfer data from one computer to another, especially from small laboratory systems, word processors, or home computers to large mainframe computers. In many of these cases, there are no common storage media; home computers do not have 9-track tape drives and large mainframes do not have 5 1/4 in. floppy disk drives. Transmission of data through the telephone network is a viable method for data transfer, which is paradoxically both easier than many believe and more difficult than some may claim. One of the keys to successful data transmission is an understanding of telecommunications protocols, i.e., the rules governing intersystem communication through the telephone network. Some of the most common protocols allow exchanging ASCII-coded data at either 300 or 1200 baud. A variety of computer systems can be used, including IBM and DEC mainframes, a Wang word processor, an IBM PC-compatible microcomputer, and the Atari 800 microcomputer. A specific example is the use of the Atari 800 as an APL terminal, complete with the custom character set, standard ASCII text, and data transfer.

Molecular dynamics simulation and NMR study of a blood group H trisaccharide.

Molecular dynamics simulations in vacuum and solution have been carried out on 2'-alpha-L-fucosyl-lactitol, a model for blood group H in conjunction with two-dimensional nmr measurements on the same compound. Three independent starting conformations for the dynamics were chosen from low energy conformations obtained by a phi/psi grid search. Nine 5 ns vacuum simulations of the trisaccharide were performed, employing three different ways to treat electrostatic interactions for each starting conformation: distance-dependent dielectric with epsilon = r, constant dielectric with epsilon = 1, or constant dielectric with epsilon = 80. In vacuum, transitions of phi and psi for the alpha-L-Fuc-(1-->2)-beta-D-Gal element occur in a cooperative manner. The virtual distance obtained for H1 in fucose to H2 in galactose from nuclear Overhauser effect spectroscopy experiments agree with one of the conformations of the trisaccharide in one of the three 100 ps aqueous simulations (phi/psi ca. -100 degrees/150 degrees), indicating this may be a dominant solution conformation. The rms fluctuations of the phi- and psi-dihedral angles were approximately 10 degrees for a conformational state, both in the vacuum and the aqueous simulations. For the simulations in vacuum, the agreement with experimental NOE data is reasonable when a constant dielectric of 1 is used (major conformers having phi/psi ca. -100 degrees/150 degrees and -140 degrees/100 degrees), whereas the agreement was poor with a constant dielectric of 80. Translational diffusion coefficients calculated from the simulation of the oligosaccharides were 0.12-0.18 x 10(-5) cm2/s and from nmr measurements 0.27 x 10(-5) cm2/s.

Theoretical studies of relaxation of a monomeric subunit of HIV-1 protease in water using molecular dynamics.

The dynamic behavior of one 99-residue subunit of the dimeric aspartyl protease of HIV-1 was studied in a 160 psec molecular dynamics simulation at 300 K in water. The crystal structure of one of the identical subunits of the dimer was the starting point, with the aqueous phase modeled by 4,331 explicit waters in a restrained spherical droplet. Analysis of the simulations showed that the monomer displayed considerable flexibility in the interfacial portions of the flap (the region which folds over the substrate), the N- and C-termini, and, to a lesser extent, the active site. The flap undergoes significant motion as an independent rigid finger, but without the cantilever previously reported in a simulation of the dimer. The N-terminus displayed the greatest fluctuational disorder whereas the C-terminus exhibited the greatest root mean square movement from the crystal structure. The central core of the monomer had a heavy-atom root mean square deviation from the initial structure of about 3.0 A during the latter half of the simulation. Although this is larger than the 1.6 A found for comparable simulations of typical globular proteins, the general features of the tertiary structure were preserved over the course of the simulation. Overall, these results indicate that the relaxed structure obtained in these simulations may provide a better model for the tertiary structure of the solvated HIV-1 protease monomer than the subunit conformation seen in the X-ray crystallographic structure of the dimer. Except in the flap region, the design of compounds intended to interfere with dimerization should take this relaxation and the flexibility of the solvated monomer, especially at the termini, into account.

A method for characterizing transition concertedness from polymer dynamics computer simulations.

A statistical method based on classifying the transitions among a set of dihedral angles within an "energy transfer window" is developed, and used to analyze Brownian (BD) and molecular dynamics (MD) simulations of the acyl chains in a lipid bilayer, and MD of neat hexadecane. It is shown for the BD simulation that when a transition of the dihedral angle in the center of the chain occurs, a transition of a particular next nearest neighbor (or angle 2-apart) will follow concertedly with a probability of approximately 0.10 within a time window of approximately 3 ps. The MD bilayer simulations, which are based on a more flexible model of the hydrocarbon chains, yield corresponding concerted transition probabilities of approximately 0.083 and window sizes of 1-2 ps. An analysis of angles 4-apart yields concerted transition probabilities of 0.03 and 0.04 for the BD and MD bilayer simulations, respectively, and window sizes close to those of the corresponding 2-apart cases. Statistical hypothesis testing very strongly rejects the assertion that these follower transitions are occurring at random. Similar analysis reveals marginal or no evidence of concertedness between 1-apart (nearest neighbor) and between 3-apart dihedral angle transitions. The pattern of concertedness for hexadecane is qualitatively similar to that of the lipid chains, although concertedness is somewhat stronger for the 3-apart transitions and somewhat weaker for those 4-apart. Finally, it is suggested that the diffusion of small solute molecules in membranes is better facilitated by nonconcerted transitions, which are associated with relatively large displacements of the chains, than by concerted transitions, which do little to change the chain shape.

Model for the structure of the lipid bilayer.

A detailed model for the structure and dynamics of the interior of the lipid bilayer in the liquid crystal phase is presented. The model includes two classes of motion: (i) the internal dynamics of the chains, determined from Brownian dynamics simulations with a continuous version of the Marcelja mean-field potential, and (ii) noncollective reorientation (axial rotation and wobble) of the entire molecule, introduced by a cone model. The basic unit of the model is a single lipid chain with field parameters adjusted to fit the 2H order parameters and the frequency-dependent 13C NMR T1 relaxation times of dipalmitoyl phosphatidylcholine bilayers. The chain configurations obtained from the trajectory are used to construct a representation of the bilayer. The resulting lipid assembly is consistent with NMR, neutron diffraction, surface area, and density data. It indicates that a high degree of chain disorder and entanglement exists in biological membranes.

Conformational states of a TT mismatch from molecular dynamics simulation of duplex d (CGCGATTCGCG).

The TT mismatch region in duplex d (CGCGATTCGCG) was studied using a 500-ps molecular dynamics (MD) simulation in water, and a series of 1-ps MD simulations and energy minimizations in vacuum. The DNA maintained its duplex structure, although the mismatch region showed significantly higher flexibility than the GC regions. The predominant conformation in the 500-ps MD simulation involved an average -42 degrees propeller twist between T6 and T'6, and a -22 degree buckle between A5 and T'7. One hydrogen bond was formed between T6 and T'6, and another between T6 and the O2 of T'7, with both Watson-Crick hydrogen bonds between A5 and T'7 remaining intact. The minimizations resulted in conformations with the equivalent hydrogen-bonding pattern, as well as ones with "wobble pair" hydrogen bonds between T6 and T'6. However, the wobble pair conformation was found to be unstable in the water simulation.

Separation and analysis of pilocarpine, isopilocarpine, and pilocarpic acid by reverse phase liquid chromatography: collaborative study.

A method for separating and determining pilocarpine and 2 degradation products was developed and subjected to collaborative study. Pilocarpine, isopilocarpine, and pilocarpic acid were isolated on a reverse phase liquid chromatographic phenyl bonded column and detected by UV spectrophotometry at 220 nm. Nine collaborators received commercial samples labeled to contain 2, 1, and 0.5% pilocarpine and a 2% practice sample. The collaborative results for pilocarpine were excellent; coefficients of variation ranged from 3.20 to 4.10%. The method was adopted official first action for determination of the active component, pilocarpine, in the presence of isopilocarpine and pilocarpic acid. Although quantitative results for the degradation products were not as good, the method is suitable as a limits test for these substances.