Developing analytical tools for saccharides in condensed phases

Carbohydrates are conformationally very complex molecules. It is this complexity that lies at the basis of the important roles that these molecules play in many biochemical and biomaterial systems. Moreover, the unusual response of these macromolecules to their environment allow them to play these often critical roles. This is particularly true for solvated carbohydrates. A knowledge of the molecular structure of carbohydrates is essential for an understanding of their function and the molecular basis of their macroscopic properties. The details of solution structure have proven difficult to probe experimentally, but computer simulations are a means for examining solvent structure directly. In this thesis we develop various computational methods for analysing saccharides in solution and in the solid state. These methods are applied to molecular dynamics simulations of maltose, hexa-amylose and a series of cyclodextrins in solution, in order to investigate the effects of water on these polysaccharides. Maltose is investigated because of its potential as a model for larger polysaccharides comprising α(1 → 4)-linked glucose monomers. Solvation was found to effect the conformations of the saccharides studied considerably. In particular, the range of motion around the glycosidic linkage is increased. Comparison of the dynamics around the glycosidic linkages for the various simulation show that oligosaccharides linked via α(1 → 4) glycosidic linkages have similar behaviour around this linkage. The saccharides studied were found to impose considerable anisotropic structure on the surrounding water which may give insights into their solution properties. In addition to the studies in solution, a recently developed method for analysing the close contacts in crystal structures is applied to crystal structures of cyclodextrin inclusion compounds. It shown to be a useful tool for investigating hydrogen-bonding patterns in the cyclodextrins

Conformational free energy maps for globobiose (a-D-Galp-(1-4)-b-D-Galp) in implicit and explict aqueous solution

Four Ramachandran maps of the conformational potential of mean force (PMF) for the galactose disaccharide globobiose(alpha-D-Galp-(1-4)-beta-D-Galp) were calculated in vacuum, explicit water, with a simple high dielectric constant and a distance dependent dielectric coefficient, respectively. This simple model of the galactanalpha-(1-4)-linkage is shown to be conformationallyrestricted, with only a small range of syn-phi syn-psi conformations predominating at standard temperature and pressure. This has implications for the preferred conformation and chain dynamics of alpha-galactosides. In addition, comparison of the relevant PMF surfaces reveals the substitution of a high dielectric constant for explicit water solution to be a valid approximation for reproducing the minimum energy conformation of this glycosidic linkage

Detection of binary pulsars with GPU-accelerated sinusoidal Hough transformations.

Analysis of relativistic binary pulsars is currently the best means by which to test theories of gravity in strong gravitational fields. Four-dimensional Hough transformations can detect sinusoids in noisy images. Hough transformations can by applied to Dynamic Power Spectra to detect the sinusoidal sift in observed spin frequency from binary pulsars in approximately circular orbits. We present four alternative GPU implementations of a Hough transformation algorithm, which we apply to synthesized Dynamic Power Spectra data to determine the GPU kernel that provides the best acceleration

Conformation and cross-protection in Group B Streptococcus serotype III and Streptococcus pneumoniae serotype 14: a molecular modeling study

Although the branched capsular polysaccharides of Streptococcus agalactiae serotype III (GBSIII PS) and Streptococcus pneumoniae serotype 14 (Pn14 PS) differ only in the addition of a terminal sialic acid on the GBSIII PS side chains, these very similar polysaccharides are immunogenically distinct. Our simulations of GBSIII PS, Pn14 PS and the unbranched backbone polysaccharide provide a conformational rationale for the different antigenic epitopes identified for these PS.We find that side chains stabilize the proximal bDGlc(1->6)bDGlcNAc backbone linkage, restricting rotation and creating a well-defined conformational epitope at the branch point. This agrees with the glycotope structure recognized by an anti-GBSIII PS functional monoclonal antibody. We find the same dominant solution conformation for GBSIII and Pn14 PS: aside from the branch point, the backbone is very flexible with a “zig-zag” conformational habit, rather than the helix previously proposed for GBSIII PS. This suggests a common strategy for bacterial evasion of the host immune system: a flexible backbone that is less perceptible to the immune system, combined with conformationally-defined branch points presenting human-mimic epitopes. This work demonstrates how small structural features such as side chains can alter the conformation of a polysaccharide by restricting rotation around backbone linkages

Effective Visualization of Tuberculosis Three-Drug Assays: A Design Study

The rise in multidrug-resistant tuberculosis means that new drugs and new drug combinations are needed to address the problems associated with current treatments. Drug screening facilities aim to identify new high quality drug compounds, or novel drug combinations, for treatment of tuberculosis. The experimental drug assay procedure produces multivariate data that is difficult to analyse and onerous to process in order to determine which drug combinations should be pursued for further development. In this design study, we have developed a visualization tool to assist with analysis and processing of this multidimensional data. The tool was developed with an iterative user-centred design process, beginning with a low fidelity paper mock-up through to the deployment of a fully functional, computer-based prototype that expert users judge to be both usable and effective

Implementation, Validation and Profiling of a Genetic Algorithm for Molecular Conformational Optimization

Prediction of the lowest energy conformation of a protein chain is a challenging optimization problem in computational chemistry and biology. Simple lattice-based protein models have been shown to be effective representations of the characteristics of proteins important in protein folding. An effective genetic algorithm for conformational optimization of proteins represented by the hydrophobic-hydrophillic lattice model was recently published. In this work, we create a publically available implementation of this genetic optimization algorithm. Tests of our implementation show equivalent performance to that reported for the original, in terms of both optimal conformation and number of function evaluations. In addition, we test our implementation across a range of data set sizes to characterize the performance of the algorithm as chain length increases: benchmarking that is necessary for future optimization and parallelization of the algorithm

Comparative simulation of pneumococcal serogroup 19 polysaccharide repeating units with two carbohydrate force fields

Streptococcus pneumoniae causes meningitis, pneumonia and severe invasive disease (IPD) in young children. Although widespread infant immunisation with the PCV7 seven-valent pneumococcal conjugate vaccine has led to a dramatic decrease in IPD, infections due to non-vaccine serotypes, particularly serotype 19A, have increased. As the 19F polysaccharide differs from 19A at a single linkage position, it was assumed that PCV7 (containing 19F) would cross-protect against 19A disease. However, vaccination with PCV7 results in only 26% effectiveness against IPD caused by 19A. We explored the conformations and dynamics of the polysaccharide repeating units from serotypes 19F and 19A, comparing free energy surfaces for glycosidic linkages with 100 ns aqueous molecular dynamics simulations of the di- and trisaccharide components. All calculations were performed with both the CHARMM and the GLYCAM carbohydrate force fields to establish whether the choice of model affects the predicted molecular behaviour. Although we identified key differences between the force fields, overall they were in agreement in predicting a 19F repeating unit with a wider range of conformation families than the more restricted 19A trisaccharide. This suggests a probable conformational difference between the 19F and 19A polysaccharides, which may explain the low cross-protection of 19F vaccines against 19A disease

Cryptococcus neoformans capsular GXM conformation and epitope presentation: a molecular modelling study

The pathogenic encapsulated Cryptococcus neoformans fungus causes serious disease in immunosuppressed hosts. The capsule, a key virulence factor, consists primarily of the glucuronoxylomannan polysaccharide (GXM) that varies in composition according to serotype. While GXM is a potential vaccine target, vaccine development has been confounded by the existence of epitopes that elicit non-protective antibodies. Although there is evidence for protective antibodies binding conformational epitopes, the secondary structure of GXM remains an unsolved problem. Here an array of molecular dynamics simulations reveal that the GXM mannan backbone is consistently extended and relatively inflexible in both C. neoformans serotypes A and D. Backbone substitution does not alter the secondary structure, but rather adds structural motifs: bDGlcA and bDXyl side chains decorate the mannan backbone in two hydrophillic fringes, with mannose-6-O-acetylation forming a hydrophobic ridge between them. This work provides mechanistic rationales for clinical observations—the importance of O-acetylation for antibody binding; the lack of binding of protective antibodies to short GXM fragments; the existence of epitopes that elicit non-protective antibodies; and the self-aggregation of GXM chains—indicating that molecular modelling can play a role in the rational design of conjugate vaccines

An eHealth Android Application for Mobile Analysis of Microplate Assays

Drug efficacy assays with microplate readers are a key aspect of the modern drug discovery process. However, microtitre plate readers are expensive laboratory equipment and not easily transportable. We have created a prototype Android smartphone application that enables smartphones to measure the concentration values in microplate wells. We find that the smartphone camera measures concentrations of red and yellow solutions more accurately than green. Further, concentration readings are most accurate when microplates are backlit and sources of noise (such as glare and shadows) are removed. Therefore, we designed a simple dark box to control ambient light, which reduces the error in measurements to within 7% of a laboratory microplate reader. An affordable and mobile alternative to a microplate reader is expected to support drug research in developing countries by enabling rapid, cheap drug assays in small laboratories and in field work with a standard Android smartphone

Conformational comparisons of Pasteurella multocida types B and E and structurally related capsular polysaccharides

Pasteurella multocida, an encapsulated gram-negative bacterium, is a significant veterinary pathogen. The P. multocida is classified into 5 serogroups (A, B, D, E, and F) based on the bacterial capsular polysaccharide (CPS), which is important for virulence. Serogroups B and E are the primary causative agents of bovine hemorrhagic septicemia that is associated with significant yearly losses of livestock worldwide, primarily in low- and middle-income countries. The P. multocida disease is currently managed by whole-cell vaccination, albeit with limited efficacy. CPS is an attractive antigen target for an improved vaccine: CPS-based vaccines have proven highly effective against human bacterial diseases and could provide longer-term protection against P. multocida. The recently elucidated CPS repeat units of serogroups B and E both comprise a N-acetyl-β-D-mannosaminuronic acid/N-acetyl-β-D-glucosamine disaccharide backbone with β-D-fructofuranose (Fruf) side chain, but differ in their glycosidic linkages, and a glycine (Gly) side chain in serogroup B. Interestingly, the Haemophilus influenzae types e and d CPS have the same backbone residues. Here, comparative modeling of P. multocida serogroups B and E and H. influenzae types e and d CPS identifies a significant impact of small structural differences on both the chain conformation and the exposed potential antibody-binding epitopes (Ep). Further, Fruf and/or Gly side chains shield the immunogenic amino-sugar CPS backbone—a possible common strategy for immune evasion in both P. multocida and H. influenzae. As the lack of common epitopes suggests limited potential for cross-reactivity, a bivalent CPS-based vaccine may be necessary to provide adequate protection against P. multocida types B and E