GABA receptor associated protein changes the electrostatic environment around the GABA type A receptor.

We have performed fully atomistic molecular dynamics simulations of the intracellular domain of a model of the GABAA receptor with and without the GABA receptor associated protein (GABARAP) bound. We have also calculated the electrostatic potential due to the receptor, in the absence and presence of GABARAP. We find that GABARAP binding changes the electrostatic properties around the GABAA receptor and could lead to increased conductivity of chloride ions through the receptor. We also find that ion motions that would result in conducting currents are observed nearly twice as often when GABARAP binds. These results are consistent with data from electrophysiological experiments.

Functional movements of the GABA type A receptor.

We have performed a parallel tempering crankshaft motion Monte Carlo simulation on a model of the GABA type A receptor with the aim of exploring a wide variety of local conformational space. We develop a novel method to analyse the protein movements in terms of a correlation tensor and use this to explore the gating process, that is, how agonist binding could cause ion channel opening. We find that simulated binding impulses to varying clusters of GABA binding site residues produce channel opening, and that equivalent impulses to single GABA sites produce partial opening.

Prediction of GABARAP interaction with the GABA type A receptor.

We have performed docking simulations on GABARAP interacting with the GABA type A receptor using SwarmDock. We have also used a novel method to study hydration sites on the surface of these two proteins; this method identifies regions around proteins where desolvation is relatively easy, and these are possible locations where proteins can bind each other. There is a high degree of consistency between the predictions of these two methods. Moreover, we have also identified binding sites on GABARAP for other proteins, and listed possible binding sites for as yet unknown proteins on both GABARAP and the GABA type A receptor intracellular domain.

Interconnected, inhabited and insecure: why bodies should not be property.

This article argues against the case for regarding bodies and parts of bodies to be property. It claims that doing so assumes an individualistic conception of the body.  It fails to acknowledge that our bodies are made up of non-human material; are unbounded; constantly changing and deeply interconnected with other bodies. It also argues that holding that our bodies are property does not recognise the fact that we have different attitudes towards different parts of our removed bodies and the contexts of their removal.  The appropriate legal reform should, therefore, be to produce a statute which can provide a balance between the competing personal, social and interpersonal interests in different body parts.

Relational bodies.

This article argues that debates over the legal status of bodies reveal a much deeper dispute over the nature of the self. In these discussions lawyers and ethicists have much to learn from a more profound understanding of the biological nature of the body. Far from being a static entity, the body is constantly recreating itself. It contains parts that are organisms in their own right. Bodies are dependent upon other bodies and the external environment for survival. The complex biological picture reflects a philosophical truth that bodies are interdependent and "leaky". We should not, therefore, expect a single legal regime, such as property to capture the biological and ethical values that are at stake in relation to every part of the body. A more complex statutory regime is required to recognise the complexity of the interests in, and nature of, different body parts.

Exploring ligand recognition and ion flow in comparative models of the human GABA type A receptor.

We present two comparative models of the GABA(A) receptor. Model 1 is based on the 4-A resolution structure of the nicotinic acetylcholine receptor from Torpedo marmorata and represents the unliganded receptor. Two agonists, GABA and muscimol, two benzodiazepines, flunitrazepam and alprazolam, together with the general anaesthetic halothane, have been docked to this model. The ion flow is also explored in model 1 by evaluating the interaction energy of a chloride ion as it traverses the extracellular, transmembrane and intracellular domains of the protein. Model 2 differs from model 1 only in the extracellular domain and represents the liganded receptor. Comparison between the two models not only allows us to explore commonalities and differences with comparative models of the nicotinic acetylcholine receptor, but also suggests possible protein sub-domain interactions with the GABA(A) receptor not previously addressed.

Exploring the binding of serotonin to the 5-HT3 receptor by density functional theory.

The 5-HT3 receptor is a typical ligand-gated ion channel of the Cys-loop superfamily, which is activated by binding of serotonin (5-HT). Models of the binding site of this protein reveal potential interactions between 5-HT and Tyr143, Tyr153, and Tyr234. Here we describe a series of ab initio calculations, based on density functional theory, to assess the effects of mutating these tyrosine residues on the binding of 5-HT. A series of mutations to these tyrosines, previously studied experimentally, were tested, and the binding energies compared with the available experimental data. Our results show that Tyr153 could form a hydrogen bond with the tertiary amine of 5-HT, and that mutation in this location revealed binding energies broadly in line with experimentally determined EC50s. Tyr143 could also form a hydrogen bond, but as EC50s do not relate to binding energies, it is unlikely that such a bond is formed here. Tyr234 is quite distinct in that it may interact with 5-HT via a mixed hydrogen bond/cation-pi interaction.

Constrained geometric simulation of the nicotinic acetylcholine receptor.

Constrained geometric simulations have been performed for the recently published closed-channel state of the nicotinic acetylcholine receptor. These simulations support the theory that correlated motion in the flexible ß-sheet structure of the extracellular domain helps to communicate a "conformational wave", spreading from the acetylcholine binding pocket. Furthermore, we have identified key residues that act at the interface between subunits and between domains that could potentially facilitate rapid communication between the binding site and the transmembrane gate.

New insights into the molecular mechanisms of general anaesthetics.

This paper provides new insights of how general anaesthetic research should be carried out in the future by an analysis of what we know, what we do not know and what we would like to know. I describe previous hypotheses on the mechanism of action of general anaesthetics (GAs) involving membranes and protein receptors. I provide the reasons why the GABA type A receptor, the NMDA receptor and the glycine receptor are strong candidates for the sites of action of GAs. I follow with a review on attempts to provide a mechanism of action, and how future research should be conducted with the help of physical and chemical methods.

Unbinding pathways of an agonist and an antagonist from the 5-HT3 receptor.

The binding sites of 5-HT3 and other Cys-loop receptors have been extensively studied, but there are no data on the entry and exit routes of ligands for these sites. Here we have used molecular dynamics simulations to predict the pathway for agonists and antagonists exiting from the 5-HT3 receptor binding site. The data suggest that the unbinding pathway follows a tunnel at the interface of two subunits, which is approximately 8 A long and terminates approximately 20 A above the membrane. The exit routes for an agonist (5-HT) and an antagonist (granisetron) were similar, with trajectories toward the membrane and outward from the ligand binding site. 5-HT appears to form many hydrogen bonds with residues in the unbinding pathway, and experiments show that mutating these residues significantly affects function. The location of the pathway is also supported by docking studies of granisetron, which show a potential binding site for granisetron on the unbinding route. We propose that leaving the binding pocket along this tunnel places the ligands close to the membrane and prevents their immediate reentry into the binding pocket. We anticipate similar exit pathways for other members of the Cys-loop receptor family.

Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding.

We have used a homology model of the extracellular domain of the 5-HT(3) receptor to dock granisetron, a 5-HT(3) receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 A from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [(3)H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.

Water movement during ligand unbinding from receptor site.

An 1-ns unbinding trajectory of retinol from the bovine serum retinol-binding protein has been obtained from molecular dynamics simulations. The behavior of water during ligand unbinding has never been studied in detail. I described a new method for defining a binding site, located the water molecules involved in the binding site, and examined their movements during unbinding. I found that there were only small changes in the binding site. During unbinding, the number of water molecules inside the binding site decreased, with some water molecules exhibiting movements similar in magnitude to bulk water, and there were rearrangements of the hydrogen bonds. This work represents the first detailed study of the behavior of water during an unbinding process.

Water and protein movements in ligand-receptor interactions.

I have recently developed a novel method `mutual repulsion' for simulating ligand unbindingfrom receptor. Combined with adiabatic switching,this method can evaluate the free energy change of unbinding. Mutualrepulsion has been applied to the bovine serum retinol-bindingprotein-retinol complex (1HBP). Large changes in amino acid configurationis observed in only three residues at the mouth of the binding site. Thechange in water structure around the ligand, from bulk-phase tohydrophobic hydration, as retinol unbinds, is also described.

Automated site-directed drug design: the generation of a basic set of fragments to be used for automated structure assembly.

If a method is to be developed to assemble putative ligand structures in site-directed drug design, from molecular graphs generated in the site, then basic building blocks are needed. Structure assembly is a combinatoric process that needs to be optimised if it is to be tractable. What has to be determined is whether small molecular fragments can have transferable properties from one molecule to another. In this paper we determine all possible combinations of 3-, 4- and 5-atom aliphatic fragments from a small set of atoms H, C, N, O, F or Cl. The frequency of occurrence of these candidate fragments is searched for in the Cambridge Structural Database. A similar analysis is performed on charged fragments. A more restricted search is carried out for P and S and aromatic structures. A basic set of fragments can be derived that have a significant frequency in known crystal structures. The transferability of fragment properties is discussed in subsequent papers.

Automated site-directed drug design: searches of the Cambridge Structural Database for bond lengths in molecular fragments to be used for automated structure assembly.

In this paper a database of small frequently occurring molecular fragments is used for the determination of fragment bond lengths from the Cambridge Structural Database. A large number of bond types are described that have not been reported previously.

Automated site-directed drug design: an assessment of the transferability of atomic residual charges (CNDO) for molecular fragments.

In this paper a database of atomic residual charges has been constructed for all the molecular fragments defined previously in a combinatorial search of the Cambridge Structural Database. The charges generated for the atoms in each fragment are compared with charges calculated for whole molecules containing those fragments. The fragment atomic charges lie within 1 S.D. of the mean for 68%, and within 2 S.D. for 91%, of the atoms whose charges were computed for whole molecules. The actual charges on any atom are strongly influenced by the adjacent connected atoms. There is a large spread of atomic residual charge within the fragments database.

Electrostatic complementarity between proteins and ligands. 2. Ligand moieties.

Drug design strategies consider factors governing intermolecular interactions to build up putative ligands. In many strategies, the ligand is constructed using fragments which are placed in the site sequentially. The optimization is then performed with each fragment. We would like to examine if this optimization strategy could generate ligands with optimal electrostatic interactions. The electrostatic complementarities between constituent moieties and the receptor site have been calculated. The whole-ligand complementarity does not appear to be the mathematical mean of the individual complementarities, nor have we found a simple relationship between the moiety and whole-ligand complementarities. The results demonstrate clearly that, using a simple model, it is very difficult to predict the electrostatic potential complementarity of the whole ligand from the complementarities of its constituent chemical moieties. This means that ligand design strategies must optimize the electrostatic complementarity globally, and not moiety by moiety.

Electrostatic complementarity between proteins and ligands. 1. Charge disposition, dielectric and interface effects.

Electrostatic interactions have always been considered an important factor governing ligand-receptor interactions. Previous work in this field has established the existence of electrostatic complementarity between the ligand and its receptor site. However, this property has not been treated rigorously, and the description remains largely qualitative. In this work, 34 data sets of high quality were chosen from the Brookhaven Protein Databank. The electrostatic complementary has been calculated between the surface potentials; complementarity is absent between adjacent or neighbouring atoms of the ligand and the receptor. There is little difference between complementarities on the total ligand surface and the interfacial region. Altering the homogeneous dielectric to distance-dependent dielectrics reduces the complementarity slightly, but does not affect the pattern of complementarity.

Electrostatic complementarity between proteins and ligands. 3. Structural basis.

Electrostatic potential complementarity between ligands and their receptor sites is evaluated by the superposition of the electrostatic potential, generated by the receptor, onto the ligand potential over the ligand van der Waals surface. We would like to examine which structural factors generate this pattern of superposition. Example studies suggest that in many ligand-protein pairs, there exist principal formal charges on each molecule, largely responsible for the electrostatic potential complementarity observed. Electrostatic potential complementarity depends on the relative disposition of these principal charges and the ligand van der Waals surface. Simple mathematical models were constructed to predict the complementarity solely from structural considerations. The essential conditions for electrostatic potential complementarity were elucidated. These can be used in ligand design strategies to obtain an electrostatically optimal ligand.

Defining, assigning and designing sex.

This article challenges the distinction the law draws between male and female. It focuses on the legal and medical treatment of intersexual people. Analysing the nature and rate of intersexuality it argues that there is a significant number of people who cannot be described as either male or female and instead exhibit a range of sexual characteristics. Until recently the law and medicine have insisted that intersexual people should be categorized as either male or female. Surgery was performed to ensure that they had the appearance assumed to be the 'norm' for a man or woman and the law followed this medical assignment of sex. Over the last couple of years the established medical practice and the legal treatment have been challenged. This article discusses the nature of these challenges and argues that there is a strong case for rejecting the traditional legal and medical approach to intersexual people. Cosmetic surgery on intersexual babies should be delayed until the individual is old enough to be able to choose their own sexual identity, which may be neither male nor female. The insistence that every person must either be male or female is no longer supportable in medical or social terms and a much wider range of sexual identities must be recognized by the law.