Electrochemical structure of the crowded cytoplasm

The current view of the cytoplasm as a 'bustling and well-organized metropolitan city' raises the issue of how physicochemical forces control the macromolecular interactions and transport of metabolites and energy in the cell. Motivated by studies on bacterial osmosensors, we argue that charged cytoplasmic macromolecules are stabilized electrostatically by their ionic atmospheres. The high cytoplasmic crowding (25-50% of cell volume) shapes the remaining cell volume (50-75%) into transient networks of electrolyte pathways and pools. The predicted 'semi-conductivity' of the electrolyte pathways guides the flow of biochemical ions throughout the cytoplasm. This metabolic and signaling current is powered by variable electrochemical gradients between the pools. The electrochemical gradients are brought about by cellular biochemical reactions and by extracellular stimuli. The cellular metabolism is thus vectorial not only across the membrane but also throughout the cytoplasm.     

Coarse-grained molecular simulation of self-assembly nanostructures of CTAB on nanoscale graphene

Coarse-grained molecular dynamics simulation has been performed to study the aggregated morphology of the cationic surfactant, cetyltrimethylammonium bromide (CTAB), adsorbed on nanoscale graphene surfaces. The CTAB surfactants can self-assemble on graphene to form various supramolecular morphologies and structures. The effect of packing density, thickness of graphene sheet and width of graphene nanoribbon on the CTAB–graphene self-assembly has been investigated. The buoyant densities of various graphene–CTAB assemblies were calculated, which increase with surfactant coverage and number of graphene layers. This result demonstrates that density gradient can be used to isolate graphenes with various layers. This simulation provides larger-scale microscopic insight into the supramolecular self-assembly nanostructures for the CTAB surfactants aggregated on graphene, which could be valuable to guide fabrication of graphene-based hybrid nanocomposites.     

Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

Macromolecular diffusion in crowded solutions.

The effects of crowding on the self or tracer diffusion of macromolecules in concentrated solutions is an important but difficult problem, for which, so far, there has been no rigorous treatment. Muramatsu and Minton suggested a simple model to calculate the diffusion coefficient of a hard sphere among other hard spheres. In this treatment, scaled particle theory is used to evaluate the probability that the target volume for a step in a random walk is free of any macromolecules. We have improved this approach by using a more appropriate target volume which also allows the calculation to be extended to the diffusion of a hard sphere among hard spherocylinders. We conclude that, to the extent that proteins can be approximated as hard particles, the hindrance of globular proteins by other proteins is reduced when the background proteins aggregate (the more so the greater the decrease in particle surface area), the hindrance due to rod-shaped background particles is reduced slightly if the rod-like particles are aligned, and the anisotropy of the diffusion of soluble proteins among cytoskeletal proteins will normally be small.     

Coarse-grained molecular dynamics simulation of interactions between cyclic lipopeptide Bacillomycin D and cell membranes

According to experimental studies, Bacillomycin D has strong antimicrobial activities, but the antimicrobial mechanism is still unknown. In this paper, the interaction mechanisms between this cyclic lipopeptide and three different charged cell membranes are studied via Coarse-Grained Molecular Dynamics (CG MD) simulations. A specific CG model for the cyclic lipopeptide Bacillomycin D was developed. The insertion of cyclic lipopeptide Bacillomycin D into DOPC, DOPC/DPPA and DOPC/DOTAP cell membranes was investigated. The position distribution and stability of Bacillomycin D in the three different cell membranes were analysed and compared based on density profile calculations. Additionally, we focused on the Radial Distribution Function (RDF) curves between amino acid residues with negative charges or strong hydrophobic properties and the head groups of two different cell membranes. Based on changes in the curvature of the three membranes, the cyclic lipopeptide Bacillomycin D can cause localised surface protrusions in DOPC/DOTAP membranes, inward depressions in the surface of DOPC/DPPA membranes and inhibition deformation in the surface of DOPC membranes. This study will help to further understand the antibacterial mechanism of the cyclic lipopeptide Bacillomycin D and provide a basis for the development of new antibiotics.     

Perturbation analysis of coupled diffusion and reaction with high-order kinetics

Asymptotic solutions for the effectiveness factor and the concentration profile are obtained for mth-order chemical reactions inside a slab catalyst pellet with Robin boundary condition at the pellet's outer surface. Using perturbation analysis in the limit of large reaction order m, the effectiveness factor and the concentration profile are explicitly determined up to O(1/m). Higher-order solutions can be obtained in a systematic way if desired.     

Coarse-grained molecular simulations of large biomolecules

Recently, we saw a dramatic increase in the number of researches that rely on coarse-grained (CG) simulations for large biomolecules. Here, first, we briefly describe recently developed and used CG models for proteins and nucleic acids. Balance between structure-based and physico-chemical terms is a key issue. We also discuss the multiscale algorithms used to derive CG parameters. Next, we comment on the dynamics used in CG simulations with an emphasis on the importance of hydrodynamic interactions. We then discuss the pros and cons of CG simulations. Finally, we overview recent exciting applications of CG simulations. Publicly available tools and software for CG simulations are also summarized.     

Coarse-grained molecular dynamics simulations of a rotating bacterial flagellum

Many types of bacteria propel themselves using elongated structures known as flagella. The bacterial flagellar filament is a relatively simple and well-studied macromolecular assembly, which assumes different helical shapes when rotated in different directions. This polymorphism enables a bacterium to switch between running and tumbling modes; however, the mechanism governing the filament polymorphism is not completely understood. Here we report a study of the bacterial flagellar filament using numerical simulations that employ a novel coarse-grained molecular dynamics method. The simulations reveal the dynamics of a half-micrometer-long flagellum segment on a timescale of tens of microseconds. Depending on the rotation direction, specific modes of filament coiling and arrangement of monomers are observed, in qualitative agreement with experimental observations of flagellar polymorphism. We find that solvent-protein interactions are likely to contribute to the polymorphic helical shapes of the filament.     

Protein diffusion in mammalian cell cytoplasm

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.     

Coarse-grained simulation studies of peptide-induced pore formation

We investigate the interactions between lipid bilayers and amphiphilic peptides using a solvent-free coarse-grained simulation technique. In our model, each lipid is represented by one hydrophilic and three hydrophobic beads. The amphiphilic peptide is modeled as a hydrophobic-hydrophilic cylinder with hydrophilic caps. We find that with increasing peptide-lipid attraction the preferred state of the peptide changes from desorbed, to adsorbed, to inserted. A single peptide with weak attraction binds on the bilayer surface, while one with strong attraction spontaneously inserts into the bilayer. We show how several peptides, which individually bind only to the bilayer surface, cooperatively insert. Furthermore, hydrophilic strips along the peptide cylinder induce the formation of multipeptide pores, whose size and morphology depend on the peptides’ overall hydrophilicity, the distribution of hydrophilic residues, and the peptide-peptide interactions. Strongly hydrophilic peptides insert less readily, but prove to be more destructive to bilayer integrity.     

Structure prediction and molecular simulation of gases diffusion pathways in hydrogenase

Although hydrogen is considered to be one of the most promising future energy sources and the technical aspects involved in using it have advanced considerably, the future supply of hydrogen from renewable sources is still unsolved. The [Fe]- hydrogenase enzymes are highly efficient H(2) catalysts found in ecologically and phylogenetically diverse microorganisms, including the photosynthetic green alga, Chlamydomonas reinhardtii. While these enzymes can occur in several forms, H(2) catalysis takes place at a unique [FeS] prosthetic group or H-cluster, located at the active site. 3D structure of the protein hydA1 hydrogenase from Chlamydomonas reinhardtti was predicted using the MODELER 8v2 software. Conserved region was depicted from the NCBI CDD Search. Template selection was done on the basis NCBI BLAST results. For single template 1FEH was used and for multiple templates 1FEH and 1HFE were used. The result of the Homology modeling was verified by uploading the file to SAVS server. On the basis of the SAVS result 3D structure predicted using single template was chosen for performing molecular simulation. For performing molecular simulation three strategies were used. First the molecular simulation of the protein was performed in solvated box containing bulk water. Then 100 H(2) molecules were randomly inserted in the solvated box and two simulations of 50 and 100 ps were performed. Similarly 100 O(2) molecules were randomly placed in the solvated box and again 50 and 100 ps simulation were performed. Energy minimization was performed before each simulation was performed. Conformations were saved after each simulation. Analysis of the gas diffusion was done on the basis of RMSD, Radius of Gyration and no. of gas molecule/ps plot.     

Multiple diffusion mechanisms due to nanostructuring in crowded environments

One of the key questions regarding intracellular diffusion is how the environment affects molecular mobility. Mostly, intracellular diffusion has been described as hindered, and the physical reasons for this behavior are: immobile barriers, molecular crowding, and binding interactions with immobile or mobile molecules. Using results from multi-photon fluorescence correlation spectroscopy, we describe how immobile barriers and crowding agents affect translational mobility. To study the hindrance produced by immobile barriers, we used sol-gels (silica nanostructures) that consist of a continuous solid phase and aqueous phase in which fluorescently tagged molecules diffuse. In the case of molecular crowding, translational mobility was assessed in increasing concentrations of 500 kDa dextran solutions. Diffusion of fluorescent tracers in both sol-gels and dextran solutions shows clear evidence of anomalous subdiffusion. In addition, data from the autocorrelation function were analyzed using the maximum entropy method as adapted to fluorescence correlation spectroscopy data and compared with the standard model that incorporates anomalous diffusion. The maximum entropy method revealed evidence of different diffusion mechanisms that had not been revealed using the anomalous diffusion model. These mechanisms likely correspond to nanostructuring in crowded environments and to the relative dimensions of the crowding agent with respect to the tracer molecule. Analysis with the maximum entropy method also revealed information about the degree of heterogeneity in the environment as reported by the behavior of diffusive molecules.     

Monte Carlo simulation of diffusion and reaction in two-dimensional cell structures.

Diffusion and reaction processes control the dynamics of many different biological systems. For example, tissue respiration can be limited by the delivery of oxygen to the cells and to the mitochondria. In this case, oxygen is small and travels quickly compared with the mitochondria, which can be considered as immobile reactive traps in the cell cytoplasm. A Monte Carlo theoretical investigation quantifying the interplay of diffusion, reaction, and structure on the reaction rate constant is reported here for diffusible particles in two-dimensional, reactive traps. The placement of traps in overlapping, nonoverlapping, and clustered spatial arrangements can have a large effect on the rate constant when the process is diffusion limited. However, under reaction-limited conditions the structure has little effect on the rate constant. For the same trap fractions and reactivities, nonoverlapping traps have the highest rate constants, overlapping traps yield intermediate rate constants, and clustered traps have the lowest rate constants. An increase in the particle diffusivity in the traps can increase the rate constant by reducing the time required by the particles to reach reactive sites. Various diffusive, reactive, and structural conditions are evaluated here, exemplifying the versatility of the Monte Carlo technique.     

Recent progress in molecular simulation methods for drug binding kinetics

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Heterogeneous anomalous diffusion of a virus in the cytoplasm of a living cell

The infection pathway of a virus in the cytoplasm of a living cell is studied from the viewpoint of diffusion theory, based on a phenomenon observed by single-molecule imaging. The cytoplasm plays the role of a medium for stochastic motion of a virus contained in an endosome as well as a free virus. It is experimentally known that the exponent of anomalous diffusion fluctuates in localized areas of the cytoplasm. Here, generalizing the fractional kinetic theory, such fluctuations are described in terms of the exponent locally distributed over the cytoplasm and a theoretical proposition is presented for its statistical form. The proposed fluctuations may be examined in an experiment of heterogeneous diffusion in the infection pathway.     

Free diffusion coefficient of ionic calcium in cytoplasm

The free diffusion coefficient of ionic Ca was measured in isolated samples of Myxicola axoplasm by following the migration of 45Ca. When precautions were taken to minimize the sequestration and chelation of 45Ca (i.e., using inhibitors, energy deprivation, and saturation of Ca chelation sites), a diffusion coefficient of 5.3 x 10(-6) cm2 s-1 was measured. The diffusion coefficient was not appreciably changed by lowering free calcium from 100 microM to approximately 10 microM or by increasing the diffusion time from ten to twenty minutes. In untreated cytoplasm taken directly from the giant axon of Myxicola, the migration of Ca was more complex and could not be described by a single diffusion coefficient. This result is interpreted to suggest that bulk movement of Ca-buffers may occur in untreated Myxicola axoplasm, a system that contains few microtubules.     

Coarse-grained molecular dynamics simulations of membrane proteins and peptides

Molecular dynamics (MD) simulations provide a valuable approach to the dynamics, structure, and stability of membrane-protein systems. Coarse-grained (CG) models, in which small groups of atoms are treated as single particles, enable extended (>100 ns) timescales to be addressed. In this study, we explore how CG-MD methods that have been developed for detergents and lipids may be extended to membrane proteins. In particular, CG-MD simulations of a number of membrane peptides and proteins are used to characterize their interactions with lipid bilayers. CG-MD is used to simulate the insertion of synthetic model membrane peptides (WALPs and LS3) into a lipid (PC) bilayer. WALP peptides insert in a transmembrane orientation, whilst the LS3 peptide adopts an interfacial location, both in agreement with experimental biophysical data. This approach is extended to a transmembrane fragment of the Vpu protein from HIV-1, and to the coat protein from fd phage. Again, simulated protein/membrane interactions are in good agreement with solid state NMR data for these proteins. CG-MD has also been applied to an M3-M4 fragment from the CFTR protein. Simulations of CFTR M3-M4 in a detergent micelle reveal formation of an alpha-helical hairpin, consistent with a variety of biophysical data. In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Finally, CG-MD simulations are extended to a more complex membrane protein, the bacterial sugar transporter LacY. Comparison of a 200 ns CG-MD simulation of LacY in a DPPC bilayer with a 50 ns atomistic simulation of the same protein in a DMPC bilayer shows that the two methods yield comparable predictions of lipid-protein interactions. Taken together, these results demonstrate the utility of CG-MD simulations for studies of membrane/protein interactions.     

New potential inhibitors of mTOR: a computational investigation integrating molecular docking,virtual screening and molecular dynamics simulation

The mTOR (mammalian or mechanistic Target Of Rapamycin), a complex metabolic pathway that involves multiple steps and regulators, is a major human metabolic pathway responsible for cell growth control in response to multiple factors and that is dysregulated in various types of cancer. The classical inhibition of the mTOR pathway is performed by rapamycin and its analogs (rapalogs). Considering that rapamycin binds to an allosteric site and performs a crucial role in the inhibition of the mTOR complex without causing the deleterious side effects common to ATP-competitive inhibitors, we employ ligand-based drug design strategies, such as virtual screening methodology, computational determination of ADME/Tox properties of selected molecules, and molecular dynamics in order to select molecules with the potential to become non-ATP-competitive inhibitors of the mTOR enzymatic complex. Our findings suggest five novel potential mTOR inhibitors, with similar or better properties than the classic inhibitor complex, rapamycin.     

Coarse-grained molecular dynamics of tetrameric transmembrane peptide bundles within a lipid bilayer

The conformations of model transmembrane peptides are studied to understand the structural and dynamical aspects of tetrameric bundles using a series of coarse grain (CG) molecular dynamics (MD) simulations since membrane proteins play a crucial role in cell function. In this work, two different amphipathic models have been constructed using similar hydrophobic/hydrophilic characteristics with two structurally distinct morphologies to evaluate the effect of roughness and hydrophilic topology on the structure of tetrameric bundles, one class that forms an ion-channel and one class that does not. Free energy calculations of typical amphipathic peptide topologies show that using a relatively smooth surface morphology allows for a stable conformation of the tetramer bundle in a diamond formation. However, the model with side chains attached to the core in order to roughen the surface has a stable square tetramer bundle which is consistent with experimental data and all-atom (AA) MD simulations. Comparisons of the CG simulations with AA MD simulations are in reasonable agreement with the formation of tetrameric homo-oligomers, partitioning within the lipid bilayer and tilt angle with respect to the bilayer normal. We concluded that a square or diamond shape tetrameric homo-oligomers could be stabilized by rational design of the peptide morphology and topology of the surface, thus allowing us to tune the permeability of the bundle or channel.