Biomedical subjects
Publications and source records attributed to Guy H Grant.
Molecular dynamics simulations have been used to investigate the dynamic behaviour of two small molecule neurotransmitter analogues in aqueous solution, leading to the elucidation of a mechanism for conformational change which is driven by the presence of the solvent molecules.
[reaction: see text] The solution-phase conformational properties of tetrameric and octameric chains of C-glycosyl alpha-d-lyxofuranose configured tetrahydrofuran amino acids (where the C-2 and C-5 substituents on the tetrahydrofuran ring are trans to each other) were examined using NMR and IR and CD in organic solvents. Studies by NMR and IR demonstrated that in chloroform solution, the tetramer 7 does not adopt a hydrogen-bonded conformation whereas the octamer 10 populates a well-defined helical secondary structure stabilized by 16-membered (i, i - 3) interresidue hydrogen bonds, similar to a pi-helix. Circular dichroism studies in trifluoroethanol are consistent with this conformation for the octamer 10, and also indicate that the tetramer 7 adopts a rigid conformation not stabilized by hydrogen bonds.
N-methyl-D-aspartate (NMDA) receptors are ligand-gated channels important in neurotransmission which are activated by the combined presence of glutamate and glycine. They are comprised of four subunits that form a dimer of dimers. The activity of NMDA receptors is modulated by a variety of endogenous ligands such as zinc ions, phenylethanolamines, polyamines and protons. Findings show that the binding sites for these modulators are found in the amino terminal domain of such receptors, but different modulators appear to affect different subunits. However, despite the enormous efforts expended in mutagenesis and patch clamp experiments on NMDA receptors, the exact assembly of these subunits and the effects of the modulatory species are not well understood. We have modelled dimers of the amino terminal domains of these receptors based on their homology with the extracellular dimer of a metabotropic glutamate receptor. Conserved cysteine residues, which have been highlighted as important in previous work, are shown to form a disulphide bridge, stabilizing a four-helix bundle between subunits. This establishes a hinge in the receptor. The model also highlights a zinc binding site in the binding crevice of the NR2a subunit of the receptor that stabilizes the open state of the amino terminal domain. The similar effect of ifenprodil is thus explained by its stabilization of the open state of the amino terminal domain (ATD). The presence of three histidine residues in the zinc site is used to explain the pH dependence of zinc inhibition. Previous work has also implicated certain residues in spermine stimulation of such receptors. The homology model shows that this site is found at the inter-subunit boundary of the dimer. This predicts a binding site between subunits, a result not calculable by the homology modelling of single subunits done previously. Finally, these results are drawn together to yield a consistent picture of NMDA receptor activation and desensitization. An understanding of how these receptors work and how they can be modulated is an important step toward rational drug design.
Ionotropic glutamate receptors (iGluRs) play important roles in neurotransmission in animals. There is growing evidence that iGluRs also play important roles in plants. Using a chemical genetics approach, which combined a pH-homeostasis mutant of Arabidopsis thaliana (de-etiolated3), several different iGluR agonists, molecular modelling, and reporter gene expression in transgenic plants, we provide evidence that iGluR agonism can induce dramatic changes in plant development and metabolism. Systematic hypothesis testing revealed a signalling circuit that integrates amino acid and sugar signals to affect elongation growth and the deposition of carbon into starch and lignins. The data show that aminoglycoside antibiotics, such as kanamycin, and polyamines impinge upon this circuit. These findings provide a mechanism for the conversion of amino acid and sugar signals into an appropriate response at the gene expression level, and underline the similarities in iGluR agonism between animals and plants.
Protein similarity estimations can be achieved using reduced dimensional representations and we describe a new application for the generation of two-dimensional maps from the three-dimensional structure. The code for the dimensionality reduction is based on the concept of pseudo-random generation of two-dimensional coordinates and Monte Carlo-like acceptance criteria for the generated coordinates. A new method for calculating protein similarity is developed by introducing a distance-dependent similarity field. Similarity of two proteins is derived from similarity field indices between amino acids based on various criteria such as hydrophobicity, residue replacement factors and conformational similarity, each showing a one factor Gaussian dependence. Results on comparisons of misfolded protein models with data sets of correctly folded structures show that discrimination between correctly folded and misfolded structures is possible. Tests were carried out on five different proteins, comparing a misfolded protein structure with members of the same topology, architecture, family and domain according to the CATH classification.
Using personal computers in a grid is permitting the in silico screening of millions of molecules to seek out potential inhibitors of agents that pose bioterror threats. Current projects are targeting anthrax and smallpox, but the approach has many attractions for investigating any known protein target and its inhibition.
Anthocyanidin synthase (ANS), an iron(II) and 2-oxoglutarate (2OG) dependent oxygenase, catalyses the penultimate step in anthocyanin biosynthesis by oxidation of the 2R,3S,4S-cis-leucoanthocyanidins. It has been believed that in vivo the products of ANS are the anthocyanidins. However, in vitro studies on ANS using optically active cis- and trans-leucocyanidin substrates identified cyanidin as only a minor product; instead both quercetin and dihydroquercetin are products with the distribution being dependent on the C-4 stereochemistry of the leucocyanidin substrates.
QM/MM methods were used to study the isomerization step from (2R)-methylmalonyl-CoA to succinyl-CoA. A pathway via a "fragmentation-recombination" mechanism is ruled out on energetic grounds. For the other radicalic pathway, involving an addition recombination step, geometries and vibrational contributions have been determined, and a barrier height of 11.70 kcal/mol was found. The effect of adjacent hydrogen-donating groups was found to reduce the energy barrier by 1-2 kcal/mol each and thus to provide a significant catalytic effect for this reaction. By means of molecular dynamics studies, the stereochemistry of the methylmalonyl-CoA mutase catalyzed reaction was examined. It is shown that TYR89 is essential for maintaining stereoselectivity of the abstraction of a hydrogen in the backreaction. The subsequent selective formation of one isomer of methylmalonyl-CoA is probably due to the presence of a bulky side chain.
An evaluation of alpha-aryl-alpha-diazodiones in tandem carbonyl ylide formation-enantioselective [3 + 2]-cycloaddition reactions is described. Such substrates were designed to allow investigation of the electronic characteristics of the dipole upon asymmetric induction. Intramolecular cycloadditions (with a tethered alkene dipolarophile) were found to occur in good to quantitative yields, with a difference in ee exhibited by the two electronically different diazodiones 8 and 9. Intermolecular cycloadditions using diazodiones 12 and 13 with DMAD and arylacetylenes 16-18 again demonstrated that electronics play a key role in determining the outcome of the cycloaddition reactions. Enantioselectivities of up to 76% were observed.
The amino acid glycine has a well-established role in signalling in the mammalian central nervous system. For example, glycine acts synergistically with the major excitatory neurotransmitter, glutamate, to regulate the influx of ions such as calcium, through N-methyl-d-aspartate (NMDA) receptors. Plants possess NMDA-like receptors, generically referred to as glutamate receptors (GLRs), named on the basis of their presumed ligand, glutamate. Previously, glycine has not been implicated in plant GLR activity or any other aspect of plant signalling. Using transgenic Arabidopsis seedlings expressing aequorin to monitor ligand-mediated changes in the cytosolic concentration of Ca2+ ([Ca2+]cyt), the data presented herein show that glutamate and glycine act synergistically to control ligand-mediated gating of calcium in plants. Glutamate and glycine synergism also regulates hypocotyl elongation. Transient increases in [Ca2+]cyt mediated by glutamate and glycine, as well as hypocotyl elongation, were inhibited by 6,7-dinitroquinoxaline-2,3 dione (DNQX), a competitive inhibitor of animal GLRs. Using a multiscale docking algorithm in combination with a molecular model of the ligand-binding domain of plant GLRs, evidence is provided indicating that glycine, and not glutamate, is likely to be the natural ligand for most plant GLR subunits. These findings uncover a hitherto unconsidered role for glycine signalling in plants, and suggest that the synergistic action of glutamate and glycine at NMDA-like receptors predates the divergence of plants and animals.
Structural genomics will yield an immense number of protein three-dimensional structures in the near future. Automated theoretical methodologies are needed to exploit this information and are likely to play a pivotal role in drug discovery. Here, we present a fully automated, efficient docking methodology that does not require any a priori knowledge about the location of the binding site or function of the protein. The method relies on a multiscale concept where we deal with a hierarchy of models generated for the potential ligand. The models are created using the k-means clustering algorithm. The method was tested on seven protein-ligand complexes. In the largest complex, human immunodeficiency virus reverse transcriptase/nevirapin, the root mean square deviation value when comparing our results to the crystal structure was 0.29 A. We demonstrate on an additional 25 protein-ligand complexes that the methodology may be applicable to high throughput docking. This work reveals three striking results. First, a ligand can be docked using a very small number of feature points. Second, when using a multiscale concept, the number of conformers that require to be generated can be significantly reduced. Third, fully flexible ligands can be treated as a small set of rigid k-means clusters.
Identification of a ligand binding site on a protein is pivotal to drug discovery. To date, no reliable and computationally feasible general approach to this problem has been published. Here we present an automated efficient method for determining binding sites on proteins for potential ligands without any a priori knowledge. Our method is based upon the multiscale concept where we deal with a hierarchy of models generated using a k-means clustering algorithm for the potential ligand. This is done in a simple approach whereby a potential ligand is represented by a growing number of feature points. At each increasing level of detail, a pruning of potential binding site is performed. A nonbonding energy function is used to score the interactions between molecules at each step. The technique was successfully employed to seven protein-ligand complexes. In the current paper we show that the algorithm considerably reduces the computational effort required to solve this problem. This approach offers real opportunities for exploiting the large number of structures that will evolve from structural genomics.
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By reducing protein structures to two-dimensional representations, it is possible to speed up the alignment of the structures and hence calculate similarity indices faster that using three-dimensional representations. Using amino acid based representations gives much better discrimination between proteins and faster calculations. Taking into account the relative similarity of the amino acids involved allowed improved accuracy at very little time cost.