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Fast synaptic transmission is crucial for real-time functioning of the brain. All the receptor molecules that mediate fast transmission events are also ligand-gated ion channels, i.e., they are ion channels that undergo allosteric structural changes on binding a particular neurotransmitter molecule, resulting in the opening of the channel, the entry of selected ions into the neuron and subsequent signalling events. Their primary function is to receive signal input at postsynaptic membranes, where some also play central roles in synaptic plasticity. However, they are also found in postsynaptic membranes outside synapses, and in presynaptic terminals, where they are involved in the control of transmitter release. This chapter presents an overview of current knowledge of the molecular biology of these receptors.

G protein-coupled receptors (GPCRs) transduce extracellular signals into a multitude of intracellular changes including changes in electrical activity, levels of second messengers, secretion, morphology, growth, and differentiation. Understanding how the common structural features of these receptors allow such controlled yet complex signalling patterns is a fundamental question in neurobiology. This chapter discusses the overall structural features, receptor-ligand interactions, receptor-G protein interactions, and regulation of G protein-coupled receptor function.

This chapter focuses on the first two elements of the group: oxygen and sulfur. There is a section each on dioxygen and ozone, oxygen and dioxygen coordination compounds, and structure of water and ices (including protonated water species). For sulfur, coverage includes its allotropes and polyatomic sulfur species, metal complexes with sulfide anions as ligands, and oxides and oxoacids of sulfur. Finally, there is a section on the allotropes and stereochemistry of selenium and tellurium, as well as the polyatomic anions and cations of these elements.

Ionotropic glutamate receptors (iGluRs) are the principal mediators of fast excitatory transmission in the central nervous system. These receptors were originally distinguished by their specific binding of and responses to agonists such as N-methyl-D-aspartate (NMDA), quisqualate/α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA), and kainate, thus defining three subfamilies. More recent molecular biological studies have basically confirmed that the principal receptor types fall into these three main classes on the basis of similarities in amino acid sequence, but they also have indicated that each subfamily comprises more than one gene and, as a result of posttranscriptional modifications, many more receptor protein subunits. In addition, researchers have identified a further subgroup in vertebrates (the orphan δ receptors, δ1 and δ2) and another subfamily, the kainate binding proteins, in non-mammalian vertebrates. This chapter discusses the general characteristics of ionotropic glutamate receptors, posttranscriptional modifications, alternative splicing, RNA editing, ligand-binding site of iGluRs, mechanism of channel gating of iGluRs, and the ion channel and carboxyl-terminal domain of glutamate receptors.

Ion channels are cellular proteins that conduct the movement of ions from one side of a membrane to the other. The resultant changes in local ion concentrations and electrical field play pivotal roles in physiological processes, as wide ranging as cell to cell communication, cell proliferation and secretion. This chapter provides a brief historical perspective then introduces the basic theory, terminology, and generic structural and functional features of ion channels. In addition, an overview of relevant ion channel methodologies is provided. The chapter aims to set the scene and to equip the non-specialist reader with sufficient background and understanding to comprehend and enjoy subsequent chapters which provide a more detailed analysis of channel families and individual channels.

This chapter discusses the detection and properties of electronic Na⁺−K⁺ transport in the squid axon membrane. The Na pump in the plasma membrane of animal cells mediates uphill transport of Na⁺ and K⁺. A number of investigators have sought experimental evidence for the Na/K pumping rate as a function of the membrane potential in a variety of cells and tissues. The occlusion of Na⁺ at the external surface is enhanced greatly with the decrease of external Na⁺ concentration and that occlusion of K⁺ is also greatly enhanced with the decrease of external K⁺ concentration. This seems to be a primary condition for the enzyme to pump in or pump out the ligand. The enzyme seems to be so designed as to adjust its structure for the change in ligand concentration and to continue its pumping function, even though its i _p -V characteristic shifts along the voltage axis. Changes of rate constants in the two state model with external K⁺ and Na⁺ concentrations are described in the chapter. If a similar mechanism is involved at the inner surface of the enzyme, changes of rate constants with internal K⁺ and Na⁺ concentrations can be anticipated. What can be expected with a decrease of internal Na⁺ concentration are a great increase of r ₂₁, almost no change of r ₁₂, decrease of k ⁰ ₁₂ and an increase of k ⁰ ₂₁. On the other hand, what can be expected with a decrease of internal K⁺ concentration are a great increase of r ₁₂, almost no change of r ₂₁, a decrease of k ⁰ ₁₂ and a great decrease of k ⁰ ₂₁. However, these remain to be proved.

NMDA receptors have a rich and diverse pharmacology reflecting the presence of multiple ligand binding sites at which agonists, antagonists, and modulators interact in an allosteric manner. This chapter describes concentration jump experiments used to characterize the activation of NMDA receptors by agonists and the block of NMDA receptor activity by competitive antagonists. These results were obtained before cDNAs for multiple families of NMDA receptor subunits were identified, and although the experiments illustrate important principles which would be expected to apply to all subtypes of NMDA receptor, a challenging task for the future will be to characterize in detail the ligand binding characteristics of native NMDA receptor subtypes in different areas of the brain.

This chapter compares the anatomical and pharmacological properties of the recombinant NMDA receptor subunits with the properties of native NMDA receptor subtypes. It first summarizes the anatomical distribution of individual receptor subunits and then compares their distributions with those of NMDA receptor subtypes identified in radio ligand binding studies. Radioligand binding studies indicate the presence of at least four pharmacologically distinct populations of NMDA receptors with differing regional distributions. There is substantial regional variation among members of both the NR1 and NR2 subunit families; however, the distributions of the NR2 subunits correspond much more closely to the distributions of the pharmacologically distinct NMDA receptor populations.

Classification of 5-hydroxytryptamine (5-HT, serotonin) receptors in the guinea pig ileum into ‘D’ receptors located on smooth muscle cells and those located on intramural cholinergic neurones of the myenteric plexus (M type) has been superseded by the classification into ‘5-HT₁-like’, 5-HT₂, and 5-HT_3. Pharmacological analysis of the 5-HT₂ receptor suggested that it was identical to Gaddum's ‘D’ receptor. The 5-HT₃ receptor appears to be the acceptor component of a ligand-gated cation channel which bears comparison with the nicotinic acetylcholine-operated ion channel. Serotonergic activation causes a rapid depolarizing response associated with an increase in the membrane conductance to Na⁺ and K⁺ ions. The explosion of interest in this receptor subtype and the plethora of new and very selective 5-HT₃ antagonists owe much of their origin to pioneer studies undertaken in the late 1970s. 2-Methyl-5-HT is a potent agonist at 5-HT₃ receptors, but it also has high affinity for 5-HT_1D receptors and appreciable affinity for 5-HT_1A and 5-HT_lc sites. Phenylbiguanide, on the other hand, appears to be a much more selective 5-HT₃ agonist. However, phenylbiguanide does cause carrier-mediated release of dopamine from slices of rat caudate which bodes caution in interpretation of its effects in intact tissues.

I shall now describe a special case of the Lewis acid–base reactions I introduced in Reaction 9. I showed there that a Lewis acid is a species that can accept a lone pair from another incoming species and form a bond to it, that a Lewis base is a species that provides that lone pair, and that the result of this sharing is a complex of the two species joined together by a chemical bond. The important special case I would like to share with you here is when the Lewis acid is a metal atom or ion, especially but not necessarily one drawn from the d-block of the periodic table (a ‘transition metal’). The d-block consists of the elements that make up the skinny central rectangle of the periodic table. They include important constructional metals, such as iron, nickel, and copper, and also the chemically aloof ‘noble’ metals gold, platinum, and silver. The Lewis base that I focus on will be a molecule or ion that also has an independent existence in the wild, such as water, H2O, or ammonia, NH3. In most cases the complex consists of the central metal atom or ion with up to six Lewis bases clustering around it. In this context, the Lewis bases are known as ‘ligands’ (from the Latin for ‘bound’) and I shall use that term here. I don’t want you to think that I am embarking on stratospherically esoteric material again. These metal complexes are hugely important in many aspects of the everyday world. For instance, chlorophyll is a complex of magnesium and is responsible for capturing the energy of the Sun for photosynthesis (Reaction 26). There is hardly a more important molecule. One that comes close in importance is haemoglobin, an elaborate complex of iron, which ensures that oxygen reaches all your cells and keeps you alive. Many pigments are complexes, so your life is decorated and made more colourful by them. Some pharmaceuticals are complexes based on platinum, so one day, perhaps even now, you might be kept alive by one of these artificial complexes.

As a polymer of many amino acids, any given protein can, in principle, adopt a huge number of configurations. In reality, however, the biologically stable protein adopts a single configuration that is stable over time. Thermodynamically, this configuration must represent a Gibbs free energy minimum. This chapter therefore explores how the thermodynamics and kinetics of protein folding and unfolding can be investigated experimentally (using, for example, chaotropes, heating or ligand interactions), and how these measurements can be used to enrich our understanding of protein configurations and stability.

Adsorption experiments involving soil particles typically are performed in a sequence of three steps: (1) reactio of an adsorptive (ion or molecule) with a soil contacting an aqueous solution of known composition under controlled temperature and applied pressure for a prescribed period of time, (2) separationof the wet soil slurry from the supernatant aqueous solution, and (3) quantitationof the ion or molecule of interest, both in the aqueous solution and in the separated soil slurry along with its entrained soil solution. The reaction step can be performed in either a closed system (batch reactor) or an open system (flow-through reactor), and it can proceed over a time period that is either relatively short (to investigate adsorption kinetics) or very long (to investigate adsorption equilibration). The separation step is similarly open to choice, with centrifugation, filtration, or gravitational settling being conventional methods to achieve separation. The quantitation step, in principle, should be designed not only to determine the moles of adsorbate and unreacted adsorptive, but also to verify whether unwanted side reactions, such as precipitation of the adsorptive or dissolution of the adsorbent, have influenced the experiment. After reaction between an adsorptive i and a soil adsorbent, the moles of i adsorbed per kilogram of dry soil is calculated with the standard equation ni ≡ niT − Mwmi where niT is the total moles of species i per kilogram dry soil in a slurry (batch process) or a soil column (flow-through process), Mw is the gravimetric water content of the slurry or soil column (measured in kilograms water per kilogram dry soil), and mi is the molality (moles per kilogram water) of species i in the supernatant solution (batch process) or effluent solution (flow-through process). Equation 8.1 defines the surface exces, ni, of an ion or molecule adsorptive that has become an adsorbate. Formally, ni is the excess number of moles of i per kilogram soil relative to its molality in the supernatant solution. As mentioned in Section 7.2, this surface excess may be a positive, zero, or negative quantity.

The electron density in transition metal complexes is of unusual interest. The chemistry of transition metal compounds is of relevance for catalysis, for solid-state properties, and for a large number of key biological processes. The importance of transition-metal-based materials needs no further mention after the discovery of the high-Tc superconducting cuprates, the properties of which depend critically on the electronic structure in the CuO2 planes. The results of theoretical calculations of systems with a large number of electrons can be ambiguous because of the approximations involved and the frequent occurrence of low-lying excited states. The X-ray charge densities provide independent evidence from a technique with very different strengths and weaknesses, and thus can make significant contributions to our understanding of the properties of transition-metal-containing molecules and solids. In inorganic and organometallic solids, the average electron concentration tends to be high. This means that absorption and extinction effects can be severe, and that the use of hard radiation and very small crystals is frequently essential. Needless to say that the advent of synchrotron radiation has been most helpful in this respect. The weaker contribution of valence electrons compared with the scattering of first-row-atom-only solids implies that great care must be taken during data collection in order to obtain reliable information on the valence electron distribution. When the field exerted by the atomic environment is not spherically symmetric, as is the case in any crystal, the degeneracy of the d-electron orbitals is lifted. In the electrostatic crystal field theory, originally developed by Bethe (1929) and Van Vleck (1932), all interactions between the transition metal atom and its ligands are treated electrostatically, and covalent bonding is neglected. Since the ligands are almost always negatively charged, electrons in orbitals pointing towards the ligands are repelled more strongly, and the corresponding orbitals will be higher in energy. The discussion is the simplest for the one d-electron case, in which d-d electron repulsions are absent.

Phosphofructokinase (PFK) from Escherichia coli is subject to allosteric regulation by phosphoenolpyruvate (PEP) and MgADP. These ligands inhibit and activate, respectively, by binding to a single allosteric binding domain and thereby altering the affinity the enzyme displays for its substrate, fructose-6-phosphate (Fru-6-P). The effect of hydrostatic pressure of up to 1.4 kbar on the binding of each of these ligands to PFK has been evaluated. This pressure range is insufficient to cause significant dissociation of the PFK tetramers. However, the logarithm of the equilibrium constant for each ligand binding to free enzyme decreases in a linear manner, and to virtually the same extent, when pressure is increased from 1 to 700 bar. Consequently, the ΔV associated with the binding of the inhibitor ligand PEP is virtually identical to the ΔV for the binding of the activator ligand MgADP or the substrate Fru-6-P, which falls within the range of 40–45 ml mol−1. The apparent ΔV for Fru-6-P binding decreases with increasing concentration of PEP until it is equal to +18 ml mol when PEP is fully saturating. Similarly, ΔV for Fru-6-P binding decreases to +26 ml mol−1 when MgADP is fully saturating. These data are interpreted as implying that both PEP and MgADP improve the “fit” of Fru-6-P to its binding domain despite the fact that the ligands have opposing effects on Fru-6-P binding affinity. Phosphofructokinase (PFK) from E. coli is a prototypical allosteric enzyme which was one of the first to be studied in depth (Blangy & Buc, 1967; Blangy et al., 1968) after Monod et al. (1965) published their famous proposal that allosteric behavior results from the concerted transition between discrete functional states of a protein (the MWC two-state model). PFK from E. coli is a tetrameric enzyme with a single allosteric binding domain that can bind either the activator MgADP or the inhibitor PEP. Under many circumstances the substrate, fructose 6-phosphate (Fru-6-P), binds to the enzyme with positive cooperativity, and the affinity and cooperativity that Fru-6-P exhibits is modulated by the allosteric ligands in classic K-type fashion.

For many years the idea that the activity of integral membrane proteins is regulated by the fluidity of the lipid matrix was popular and appeared to be quite rational. However, as information about the effect of viscosity on the function of different membrane proteins became available, the correlation between the two became increasingly unclear. The purpose of this article is to readdress this issue in light of our recent pressure and temperature studies. This chapter is divided into seven parts: (1) the effect of viscosity on enzyme activity; (2) the effect of viscosity on the local motions of proteins; (3) characterization of membrane viscosity; (4) demonstration of changes in protein-lipid contacts brought about by changes in viscosity; (5) an example of a protein in which the viscosity appears to stabilize a particular conformational state: (6) relations between membrane viscosity and protein function; and (7) conclusions. The effect of viscosity (η) on the rate (k) of a chemical reaction was first given by Kramers (1940): . . . k=A/ηe−Ea/RT (1) . . . In this expression, viscosity will affect the rate of a reaction by limiting the rate of diffusion of reactants. Viscosity will thus modify the frequency factor (A) and should not affect the activation energy. This expression has been applied to aqueous soluble enzymes (for example, Gavish, 1979; Gavish & Werber, 1979; Somogyi et al., 1984), and it appears that, in general, enzymes obey Kramers’s relation, although in some cases the exponent of η is less than one. Viscosity can affect enzymatic rates not only by limiting the diffusion of substrates but also by damping internal motions of the protein chains. It seems reasonable that a high enough viscosities, the protein would be damped sufficiently so that large activation energies will be required for the backbone motions that allow substrates and products to diffuse into and out of the active site. This viscosity-induced increase in activation energy was shown by studies of the reassociation of carbon monoxide and dioxygen to the heme site of myoglobin after flash photodissociation (Austin et al., 1975; Beece et al., 1980).

The question of molecular recognition is a central paradigm of molecular biology, playing central roles in most, if not all, cellular processes. Failed recognition events have been implicated in numerous disease states, ranging from flawed control of gene regulation and cellular proliferation to defects in specific metabolic activities. Historically, questions of molecular recognition have been approached through organic synthesis and through actual structural studies of biomolecular complexes. Fundamental insight into the mechanisms of molecular recognition can be realized through the use of broad interdisciplinary tools and techniques. In particular, the use of recombinant DNA technology in concert with hydrostatic and osmotic pressure methodologies have proven to be ideal for understanding the fundamental mechanisms of recognition. In our presentation, we will focus on recent results from our laboratory that examine three major classes of recognition events in biological systems: 1. Protein-protein recognition: here we seek to define the role of specific surface interactions; electrostatic, hydrogen bonding, and hydrophobic free energies provided through surface complimentarity, which define the specificity and affinity in the formation of complexes between the metalloproteins involved in electron transfer events in cytochrome P-450-dependent oxygenase catalysis and in the assembly of tetrameric hemoglobin. 2. Protein–small molecule recognition: here we seek to ascertain how the same fundamental forces of electrostatics, hydrogen bonding, and the hand-glove fit of a substrate into the active site of an enzyme can give rise to the observed high degree of control of regio- and stereo-specificity in catalysis and in the interfadal interactions of proteins at electrode interfaces. 3. Protein nucleic acid recognition: here again the same fundamental forces control recognition processes, but in this case we will focus on our exciting, recent discovery of a role for solvent water in mediating recognition between protein and nucleic acid components. Representative systems in the binding/ catalytic class of restriction endonucleases and recombinases will be discussed. In all cases, the use of pressure as a variable has provided unique understanding for the molecular details of these processes. Pressure, both hydrostatic and osmotic, has proven to be an enabling experimental technique in understanding the mechanistic origins of molecular recognition events.

The effect of hydrostatic pressure on the secondary structure of proteins can be followed by Fourier transform infrared (FTIR) spectroscopy in the diamond anvil cell. Pressure-induced changes in the amide I’ region of the deconvolved spectrum are used to follow the features of the secondary structure up to 20 kbar. The changes in the side chains such as tyrosine also can be followed. A self-deconvolution and fitting procedure is presented that allows the determination of both pressure-induced and temperature-induced changes in the secondary structure of proteins. The method takes into account the elastic, as well as the possible conformational, effects on the spectral bands of the protein. Applications are presented on pressure-induced changes in several proteins. Attention is also given to the influence of inert cosolvents. The fundamental principles of the phase diagram of proteins are presented to clarify their importance for understanding the behavior of proteins under pressure at different temperatures. Our results show that the infrared technique explores unique aspects of the behavior of proteins under these extreme conditions. The study of the effects of pressure has received considerable attention in recent years (Balny et al., 1992; Silva & Weber, 1993). In general, low pressures induce reversible changes such as the dissociation of protein-protein complexes, the binding of ligands, and conformational changes. Pressures higher than about 5 kbar induce denaturation, which in most cases is irreversible. However, reports on a few proteins indicate that such high pressures may also cause reversible changes. One such protein is horse scrum albumin (Chen & Heremans, 1990). A molecular interpretation of these phenomenon is based on the fact that pressure mainly affects the volume of a system, thus damping the molecular fluctuations. Temperature effects are known to affect both the kinetic energy and the volume of the system. Early in this century, it was shown that one can cook an egg by subjecting it to high pressure (Bridgman, 1914). The appearance of the pressure-induced coagulum of egg white is quite different from the coagulum induced by temperature.

In most undergraduate chemistry classes, students are taught to consider reactions in which cations and anions dissolved in water are depicted as isolated ions. For example, the magnesium ion is depicted as Mg2+, or at best Mg2+(aq). For anions, these descriptions may be adequate (if not accurate). However, for cations, these abbreviations almost always fail to describe the critical chemical attributes of the dissolved species. A much more meaningful description of Mg2+ dissolved in water is [Mg(H2O)6]2+, because Mg2+ in water does not behave like a bare Mg2+ ion, nor do the waters coordinated to the Mg2+ behave anything like water molecules in the bulk fluid. In many respects, the [Mg(H2O)6]2+ ion acts like a dissolved molecular species. In this chapter, we discuss the simple solvation of anions and cations as a prelude to exploring more complex reactions of soluble oxide precursors called hydrolysis products. The two key classes of water–oxide reactions introduced here are acid–base and ligand exchange. First, consider how simple anions modify the structure and properties of water. As discussed in Chapter 3, water is a dynamic and highly fluxional “oxide” containing transient rings and clusters based on tetrahedral oxygen anions held together by linear hydrogen bonds. Simple halide ions can insert into this structure by occupying sites that would normally be occupied by other water molecules because they have radii (ranging from 0.13 to 0.22 nm in the series from F- to I-) that are comparable to that of the O2- ion (0.14 nm). Such substitution is clearly seen in the structures of ionic clathrate hydrates, where the anion can replace one and sometimes even two water molecules. Larger anions can also replace water molecules within clathrate hydrate cages. For example, carboxylate hydrate structures incorporate the carboxylate group within the water framework whereas the hydrophobic hydrocarbon “tails” occupy a cavity within the water framework, as in methane hydrate (see Chapter 3). Water molecules form hydrogen bonds to dissolved halide ions just as they can to other water molecules, as designated by OH-Y-.

In Chapters 4 and 5, we demonstrated that local structures and charge distributions have an enormous impact on the equilibrium constants, trajectories, and kinetics of reactions involving soluble oxide precursors. In this chapter, we highlight those features that make reactions on extended oxide surfaces either similar to or dramatically different from the reactions documented in hydrolysis diagrams for each metal cation (see Chapter 5). We first describe oxide surface structures and then discuss how these structures impact both acid–base and ligand-exchange phenomena. In addition to dense oxides, we also introduce some of the chemistry associated with layered materials. Lamellar materials are important from both a fundamental and technological perspective, because water and ions can readily penetrate such structures and provide conditions under which almost every oxygen anion is at an oxide–water interface (see Chapter 10 and Chapter 11). This chapter focuses on oxides containing octahedral cations. The distinctive chemistry of oxides based on tetrahedral cations, including the clay minerals and the zeolites, are the focus of Part Five. The structures of bulk oxides were introduced in Chapter 2. However, for many oxides, the surface structures that interact with aqueous solutions are substantially different from structures found in the bulk. Here, we introduce the basic principles of oxide surfaces that make them chemically active. As a starting point, consider ideal oxide surfaces containing +2 octahedral cations. Pristine oxide surfaces can be created by cleaving perfect crystals in an ultrahigh-vacuum environment. The creation of new surfaces requires an expenditure of energy corresponding to the cohesive energy of the solid, which in turn represents the energy required to break every bond along a given fracture plane. For MgO, the Mg-O bond energy is 380 kJ/mole. Each surface created contains 1.4.1019 oxygen atoms/m2, or 2.4.10-5 moles of bonds. Because two surfaces are created in the fracture event, the initial interfacial energy of each resulting MgO surface is (1/2)(380 kJ/mole)/(2.4_10-5 mole/m2 )=4560 mJ/m2.

Biochemical reactions, such as substrate or coenzyme binding to enzymes are usually completed in no more than 50-100 ms and thus require rapid reaction techniques such as stopped-flow instrumentation for their study. Fortunately, many such reactions can be followed by changes in the absorption properties of the substrate, product or coenzyme, and examples of these have been described in Chapters 1, 7 and 8. An alternative possibility is that during the reaction there is a change in the fluorescence properties of the substrate, coenzyme or the protein itself. Some reactions, particularly those involving the oxidation/ reduction of coenzymes, involve both changes in absorption and changes in fluorescence emission intensity. In many cases, the fluorescence properties of the ligand or protein itself may change when a complex is formed, even in the absence of a full catalytic reaction occurring, e.g. the protein fluorescence emission of most pyridine or flavin nucleotide-dependent dehydrogenases is quenched when NAD(P)H or FADH (respectively) binds to them, due to resonance energy transfer from the aromatic amino acids of the protein to the coenzyme. Conversely, the fluorescence emission from the reduced-coenzymes is usually enhanced on formation of the complex with these enzymes (1-3). The principles behind both fluorescence and stopped-flow techniques have been described in preceding chapters (2 and 8, respectively) and therefore readers should familiarize themselves with these chapters for some of the background information. In this chapter, we discuss the use of stopped-flow fluorescence spectroscopy and its application to a number of biochemical problems. A typical stopped-flow system is assembled from modular components of a conventional spectrophotometer/fluorimeter, a device permitting rapid mixing of the components of a reaction and a data recording system with a fast response. Commercially available instruments offer facilities for the observation of changes in absorption and/or fluorescence emission after rapid mixing of the reagents. These measurements can often be made simultaneously due to the different optical requirements of the two spectroscopic techniques. Figure 1 gives a generalized diagram of the geometry of a stopped-flow system able to simultaneously measure changes in absorption and fluorescence intensity of a reaction.