Toward the atomistic simulation of T cell epitopes automated construction of MHC:peptide structures for free energy calculations

Epitopes mediated by T cells lie at the heart of the adaptive immune response and form the essential nucleus of anti-tumour peptide or epitope-based vaccines. Antigenic T cell epitopes are mediated by major histocompatibility complex (MHC) molecules, which present them to T cell receptors. Calculating the affinity between a given MHC molecule and an antigenic peptide using experimental approaches is both difficult and time consuming, thus various computational methods have been developed for this purpose. A server has been developed to allow a structural approach to the problem by generating specific MHC:peptide complex structures and providing configuration files to run molecular modelling simulations upon them. A system has been produced which allows the automated construction of MHC:peptide structure files and the corresponding configuration files required to execute a molecular dynamics simulation using NAMD. The system has been made available through a web-based front end and stand-alone scripts. Previous attempts at structural prediction of MHC:peptide affinity have been limited due to the paucity of structures and the computational expense in running large scale molecular dynamics simulations. The MHCsim server (http://igrid-ext.cryst.bbk.ac.uk/MHCsim) allows the user to rapidly generate any desired MHC:peptide complex and will facilitate molecular modelling simulation of MHC complexes on an unprecedented scale.

Ion interactions with the carbon nanotube surface in aqueous solutions:understanding the molecular mechanisms

We study the molecular mechanisms of alkali halide ion interactions with the single-wall carbon nanotube surface in water by means of fully atomistic molecular dynamics simulations. We focus on the basic physical-chemical principles of ion–nanotube interactions in aqueous solutions and discuss them in light of recent experimental findings on selective ion effects on carbon nanotubes.

Direct measurement of coherency limits for strain relaxation in heteroepitaxial core/shell nanowires

The growth of heteroepitaxially strained semiconductors at the nanoscale enables tailoring of material properties for enhanced device performance. For core/shell nanowires (NWs), theoretical predictions of the coherency limits and the implications they carry remain uncertain without proper identification of the mechanisms by which strains relax. We present here for the Ge/Si core/shell NW system the first experimental measurement of critical shell thickness for strain relaxation in a semiconductor NW heterostructure and the identification of the relaxation mechanisms. Axial and tangential strain relief is initiated by the formation of periodic a/2 〈110〉 perfect dislocations via nucleation and glide on {111} slip-planes. Glide of dislocation segments is directly confirmed by real-time in situ transmission electron microscope observations and by dislocation dynamics simulations. Further shell growth leads to roughening and grain formation which provides additional strain relief. As a consequence of core/shell strain sharing in NWs, a 16 nm radius Ge NW with a 3 nm Si shell is shown to accommodate 3% coherent strain at equilibrium, a factor of 3 increase over the 1 nm equilibrium critical thickness for planar Si/Ge heteroepitaxial growth. © 2012 American Chemical Society.

The antimicrobial action of pseudin-2

The amphibian antimicrobial peptide pseudin-2 is a peptide derived from the skin of the South-American frog Pseudis paradoxa (Olson et al., 2001). This peptide possesses tremendous potential as a therapeutic lead since it has been shown to possess both antimicrobial as well insulin-releasing properties (Olson et al., 2001; Abdel-Wahab et al., 2008). This study aimed to develop pseudin-2’s potential by understanding and improving its properties as an antimicrobial agent. The structure-function relationships of pseudin-2 were explored using a combination of in-vitro and in-silico techniques, with an aim to predict how the structure of the peptide may be altered in order to improve its efficacy. A library of pseudin-2 mutants was generated by randomizing codons at positions 10, 14 and 18 of a synthetic gene, using NNK saturation mutagenesis. Analysis of these novel peptides broadly confirmed, in line with literature precedent, that anti-microbial activity increases with increased positive charge. Specifically, 2 positively-charged residues at positions 10 and 14 and a hydrophobic at position 18 are preferred. However, substitution at position 14 with some polar, non-charged residues also created peptides with antimicrobial activity. Interestingly, the pseudin-2 analogue [10-E, 14-Q, 18-L] which is identical to pseudin-2, except that the residues at positions 10 and 14 are switched, showed no anti-microbial activity at all. Molecular dynamics simulations of pseudin-2 showed that the peptide possesses two equilibrium structures in a membrane environment: a linear and a kinked a-helix which both embed into the membrane at an angle. Biophysical characterization using circular dichroism spectroscopy confirmed that the peptide is helical within the membrane environment whilst linear dichroism established that the peptide has no defined orientation within the membrane. Collectively, these data indicate that Pseudin-2 exerts its antimicrobial activity via the carpet model.

Structure of glassy GeO2

The full set of partial structure factors for glassy germania, or GeO2, were accurately measured by using the method of isotopic substitution in neutron diffraction in order to elucidate the nature of the pair correlations for this archetypal strong glass former. The results show that the basic tetrahedral Ge(O-1/2)(4) building blocks share corners with a mean inter-tetrahedral Ge-O-Ge bond angle of 132(2)degrees. The topological and chemical ordering in the resultant network displays two characteristic length scales at distances greater than the nearest neighbour. One of these describes the intermediate range order, and manifests itself by the appearance of a first sharp diffraction peak in the measured diffraction patterns at a scattering vector k(FSDP) approximate to 1.53 angstrom(-1), while the other describes so-called extended range order, and is associated with the principal peak at k(PP) = 2.66( 1) angstrom(-1). We find that there is an interplay between the relative importance of the ordering on these length scales for tetrahedral network forming glasses that is dominated by the extended range ordering with increasing glass fragility. The measured partial structure factors for glassy GeO2 are used to reproduce the total structure factor measured by using high energy x-ray diffraction and the experimental results are also compared to those obtained by using classical and first principles molecular dynamics simulations.

Structure of molten TbCl3 measured by neutron diffraction

The total structure factor of molten TbCl3 at 617ºC was measured by using neutron diffraction. The data are in agreement with results from previous experimental work but the use of a diffractometer having an extended reciprocal-space measurement window leads to improved resolution in real space. Significant discrepancies with the results obtained from recent molecular dynamics simulations carried out using a polarizable ion model, in which the interaction potentials were optimized to enhance agreement with previous diffraction data, are thereby highlighted. It is hence shown that there is considerable scope for the development of this model for TbCl3 and for other trivalent metal halide systems spanning a wide range of ion size ratios.

Visualising and controlling the flow in biomolecular systems at and between multiple scales:from atoms to hydrodynamics at different locations in time and space

A novel framework for modelling biomolecular systems at multiple scales in space and time simultaneously is described. The atomistic molecular dynamics representation is smoothly connected with a statistical continuum hydrodynamics description. The system behaves correctly at the limits of pure molecular dynamics (hydrodynamics) and at the intermediate regimes when the atoms move partly as atomistic particles, and at the same time follow the hydrodynamic flows. The corresponding contributions are controlled by a parameter, which is defined as an arbitrary function of space and time, thus, allowing an effective separation of the atomistic 'core' and continuum 'environment'. To fill the scale gap between the atomistic and the continuum representations our special purpose computer for molecular dynamics, MDGRAPE-4, as well as GPU-based computing were used for developing the framework. These hardware developments also include interactive molecular dynamics simulations that allow intervention of the modelling through force-feedback devices.

Molecular mechanisms of salt effects on carbon nanotube dispersions in an organic solvent (N-methyl-2-pyrrolidone)

We consider the effects of salt (sodium iodide) on pristine carbon nanotube (CNT) dispersions in an organic solvent, N-methyl-2-pyrrolidone (NMP). We investigate the molecular-scale mechanisms of ion interactions with the nanotube surface and we show how the microscopic ion-surface interactions affect the stability of CNT dispersions in NMP. In our study we use a combination of fully atomistic Molecular Dynamics simulations of sodium and iodide ions at the CNT-NMP interface with direct experiments on the CNT dispersions. In the experiments we analyze the effects of salt on the stability of the dispersions by photoluminescence (PL) and optical absorption spectroscopy of the samples as well as by visual inspection. By fully atomistic Molecular Dynamics simulations we investigate the molecular-scale mechanisms of sodium and iodide ion interactions with the nanotube surface. Our simulations reveal that both ions are depleted from the CNT surface in the CNT-NMP dispersions mainly due to the two reasons: (1) there is a high energy penalty for the ion partial desolvation at the CNT surface; (2) NMP molecules form a dense solvation layer at the CNT surface that prevents ions to come close to the CNT surface. As a result, an increase of the salt concentration increases the "osmotic" stress in the CNT-NMP system and, thus, decreases the stability of the CNT dispersions in NMP. Direct experiments confirm the simulation results: addition of NaI salt into the NMP dispersions of pristine CNTs leads to precipitation of CNTs (bundle formation) even at very small salt concentration (∼10 -3 mol L -1). In line with the simulation predictions, the effect increases with the increase of the salt concentration. Overall, our results show that dissolved salt ions have strong effects on the stability of CNT dispersions. Therefore, it is possible to stimulate the bundle formation in the CNT-NMP dispersions and regulate the overall concentration of nanotubes in the dispersions by changing the NaI concentration in the solvent. © 2012 The Royal Society of Chemistry.

Structural determinants of oligomerization of the aquaporin-4 channel

The aquaporins (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3 and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family.

An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor conformational states, explaining the pharmacological preferences of calcitonin receptor-RAMP complexes. This provides novel insight into our understanding of G protein-coupled receptor-protein interaction that is likely broadly applicable for this receptor class.

Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors

The calcitonin gene-related peptide (CGRP) family of G protein- coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in aGαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMPdependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.