CHAPTER 10. Tungsten-Containing Enzymes

Coarse-grained molecular simulation models have provided immense, often general, insight into the complex behavior of condensed-phase systems but suffer from a lost connection to the true dynamical properties of the underlying system. In general, the physics that is built into a model shapes the free-energy landscape, restricting the attainable static and kinetic properties. In this work, we perform a detailed investigation into the property interrelationships resulting from these restrictions, for a representative system of the helix-coil transition. Inspired by high-throughput studies, we systematically vary force-field parameters and monitor their structural, kinetic, and thermodynamic properties. The focus of our investigation is a simple coarse-grained model, which accurately represents the underlying structural ensemble, i.e., effectively avoids sterically-forbidden configurations. As a result of this built-in physics, we observe a rather large restriction in the topology of the networks characterizing the simulation kinetics. When screening across force-field parameters, we find that structurally accurate models also best reproduce the kinetics, suggesting structural-kinetic relationships for these models. Additionally, an investigation into thermodynamic properties reveals a link between the cooperativity of the transition and the network topology at a single reference temperature.

Tauopathies are a class of neurodegenerative disorders characterized by the accumulation of tau protein filaments in the brain. On the basis of isoforms with three or four microtubule-binding repeats (3R or 4R) that constitute tau filaments, tauopathies can be divided into 3R, 4R, and 3R/4R tauopathies. [18F]PI-2620 is a tau-positron emission tomography (PET) tracer that detects tau filaments in the 3R/4R tauopathy Alzheimer's disease (AD) and the 4R tauopathies corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) with differential binding characteristics. A multiscale simulation workflow, including molecular docking, molecular dynamics simulation, metadynamics, and Brownian dynamics, was applied to uncover the molecular basis for the different binding properties of [18F]PI-2620 in these tauopathies. The energetically best binding sites of [18F]PI-2620 in the AD-tau filament are located in the C-shaped groove of the filament core structure that is accessible to the outside. The most favorable binding sites in CBD-tau and PSP-tau filaments are localized to cavities in the inner filament core. Sites on the outer surface have higher binding free energies, and interaction of [18F]PI-2620 at these sites was short-lived in the molecular dynamics simulations. Computationally predicted associated rates of [18F]PI-2620 with the groove sites in the AD-tau filament were higher than association rates with the cavity sites in the CBD- and PSP-tau filaments. The results indicate that tau filaments in AD combine favorable energetic and kinetic properties with regard to tracer binding, while the binding of [18F]PI-2620 to filaments in CBD and PSP is kinetically restricted. Our findings reveal that distinct structural, energetic, and kinetic properties of tau filaments from AD, CBD, and PSP govern their interaction with PET tracers, which highlights the possibility to achieve tau isoform specificity in future tracer developments.

The phase behavior of ternary blends was analyzed on the basis of the lattice approach. Both compatibilization and incompatibilization effects are predicted to occur depending on the relative magnitudes and the sign of the interaction parameters of the binary subsystems. Thermodynamic, structural and kinetic properties were investigated for a ternary model blend composed of poly(vinylidene fluoride), poly(methyl methacrylate) and poly(vinyl acetate). This particular ternary system is characterized by a specific symmertry with respect to the interactions in the binary subsystems. This symmetry affects both thermodynamic and structural properties. The experimentally determined interaction parameters were used to model the phase diagram on the basis of the lattice model: the theoretical phase diagram was found to be close to the experimental one. The crystallization processes were analyzed both for the binary and the ternary systems on the basis of a modified Turnbull–Fisher equation. The conclusions are that the properties of the ternary systems can be understood to a first approximation on the basis of those of the corresponding binary systems and the symmetry of the interactions.

Mixed function oxidation and enzymes: Kinetic and structural properties of an oxidatively modified alkaline phosphatase

The prevalence of Mg(2+) ions in biology and their essential role in nucleic acid structure and function has motivated the development of various Mg(2+) ion models for use in molecular simulations. Currently, the most widely used models in biomolecular simulations represent a nonbonded metal ion as an ion-centered point charge surrounded by a nonelectrostatic pairwise potential that takes into account dispersion interactions and exchange effects that give rise to the ion's excluded volume. One strategy toward developing improved models for biomolecular simulations is to first identify a Mg(2+) model that is consistent with the simulation force fields that closely reproduces a range of properties in aqueous solution, and then, in a second step, balance the ion-water and ion-solute interactions by tuning parameters in a pairwise fashion where necessary. The present work addresses the first step in which we compare 17 different nonbonded single-site Mg(2+) ion models with respect to their ability to simultaneously reproduce structural, thermodynamic, kinetic and mass transport properties in aqueous solution. None of the models based on a 12-6 nonelectrostatic nonbonded potential was able to reproduce the experimental radial distribution function, solvation free energy, exchange barrier and diffusion constant. The models based on a 12-6-4 potential offered improvement, and one model in particular, in conjunction with the SPC/E water model, performed exceptionally well for all properties. The results reported here establish useful benchmark calculations for Mg(2+) ion models that provide insight into the origin of the behavior in aqueous solution, and may aid in the development of next-generation models that target specific binding sites in biomolecules.

Models of structure and properties of liquid mixtures have been outlined. Main attention is given to the extended quasichemical approach for modeling supramolecular ordering in mixtures, selforganized by H-bonds and unified description of their physicochemical properties. Models of polyvariable supramolecular species as regard to structure and composition, taking in account the cooperativity on H-bonding, as well as the methods for describing their structure, composition, electric (dipole moment), and optic (polarisability) properties are developed. Interrelations between thermodynamic functions (Gibbs energy, enthalpy, entropy), dielectric (permittivity), and optic (refractive index and its fluctuation derivatives, determining Rayleigh light scattering) properties of mixtures, and microscopic characteristics of aggregates are analyzed. The methods for obtaining both the integral and differential parameters of aggregation are developed, applicable for structural study of the long range molecular correlations, including supramolecular aggregates of nanosizes. Models of the media with internal parameters of different nature and tensor dimension have been outlined. Backgrounds for their application to study dynamic processes of the supramolecular reorganization, intramolecular transitions, and energy transfer, as well as fluctuation and relaxation phenomena are considered. Fluctuation and relaxation contributions of internal parameters to both equilibrium and kinetic properties and Rayleigh ratio of mixtures were established. The applications to individual liquids and mixtures are illustrated. New data on the structure of aggregates and thermodynamics of their formation were obtained. Supramolecular assemblies in liquids with the long range molecular correlations were revealed. Macroscopic manifestations of the supramolecular organization in physicochemical properties of liquids are discussed.

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP(+), and Mg(2+) were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K(m) = 58 micromolar), but also inhibition by high substrate (K(i) = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K(m) = 120 micromolar). At both pH conditions, activity response to NADP(+) exhibited Michaelis-Menten behavior with K(m) values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg(2+) suggested the presence of at least two Mg(2+) - binding sites with different affinity. The K(a) values for Mg(2+) obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg(2+) levels could be important for the physiological regulation of NADP-malic enzyme.

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.

This study investigates the kinetic properties of both symmetrical and asymmetrical multilayer and nano-sized semiconductor structures. We develop a theoretical framework using various models and mathematical methods to solve the Schrödinger matrix equation for a system of electrons, taking into account the Bastard condition, which considers the difference in the effective masses of current carriers in adjacent layers. We analyze tunnel-coupled electronic states in quantum wells separated by a narrow tunnel-transparent potential barrier. Our findings provide insights into the electronic properties of semiconductor structures, which are crucial for applications in micro- or nanoelectronics and other areas of solid-state physics.

The crystal and electronic structures, thermodynamic, kinetic, energy and magnetic properties of the thermoelectric material Lu1-xZrxNiSb in the ranges: T=80–400 K, x=0–0.10 were studied. Mechanisms of simultaneous generation of structural defects of acceptor and donor nature are established. It is shown that in the structure of the basic compound LuNiSb there are defects of acceptor nature as a result of vacancies in the crystallographic positions 4a and 4c of Lu and Ni atoms, respectively, which gave rise to acceptor levels (zones) in the band gap εg. Introduction of Zr impurity atoms into the structure of the LuNiSb compound by substitution of Lu atoms in position 4a generates structural defects of donor nature with simultaneous elimination of vacancies in positions 4a and 4c of Lu and Ni atoms, respectively (acceptor levels). The ratio of the concentrations of the available defects of donor and acceptor nature determines the location of the Fermi level εF and the conduction mechanisms in Lu1-xZrxNiSb. The investigated solid solution Lu1-xZrxNiSb is a promising thermoelectric material. Bibl. 15, Fig. 9.

Substitution of the S4 of Shaw into Shaker alters cooperativity in channel activation by slowing a cooperative transition late in the activation pathway. To determine the amino acids responsible for the functional changes in Shaw S4, we created several mutants by substituting amino acids from Shaw S4 into Shaker. The S4 amino acid sequences of Shaker and Shaw S4 differ at 11 positions. Simultaneous substitution of just three noncharged residues from Shaw S4 into Shaker (V369I, I372L, S376T; ILT) reproduces the kinetic and voltage-dependent properties of Shaw S4 channel activation. These substitutions cause very small changes in the structural and chemical properties of the amino acid side chains. In contrast, substituting the positively charged basic residues in the S4 of Shaker with neutral or negative residues from the S4 of Shaw S4 does not reproduce the shallow voltage dependence or other properties of Shaw S4 opening. Macroscopic ionic currents for ILT could be fit by modifying a single set of transitions in a model for Shaker channel gating (Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. J. Gen. Physiol. 103:321–362). Changing the rate and voltage dependence of a final cooperative step in activation successfully reproduces the kinetic, steady state, and voltage-dependent properties of ILT ionic currents. Consistent with the model, ILT gating currents activate at negative voltages where the channel does not open and, at more positive voltages, they precede the ionic currents, confirming the existence of voltage-dependent transitions between closed states in the activation pathway. Of the three substitutions in ILT, the I372L substitution is primarily responsible for the changes in cooperativity and voltage dependence. These results suggest that noncharged residues in the S4 play a crucial role in Shaker potassium channel gating and that small steric changes in these residues can lead to large changes in cooperativity within the channel protein.

Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia

The present dissertation deals with the modeling of zinc oxide on the atomic scale employing both quantum mechanical as well as atomistic methods. The first part describes quantum mechanical calculations based on density functional theory of intrinsic point defects in ZnO. To begin with, the geometric and electronic structure of vacancies and oxygen interstitials is explored. In equilibrium oxygen interstitials are found to adopt dumbbell and split interstitial configurations in positive and negative charge states, respectively. Semi-empirical self-interaction corrections allow to improve the agreement between the experimental and the calculated band structure significantly; errors due to the limited size of the supercells can be corrected by employing finite-size scaling. The effect of both band structure corrections and finite-size scaling on defect formation enthalpies and transition levels is explored. Finally, transition paths and barriers for the migration of zinc as well as oxygen vacancies and interstitials are determined. The results allow to interpret diffusion experiments and provide a consistent basis for developing models for device simulation. In the second part an interatomic potential for zinc oxide is derived. To this end, the Pontifix computer code is developed which allows to fit analytic bond-order potentials. The code is subsequently employed to obtain interatomic potentials for Zn-O, Zn-Zn, and O-O interactions. To demonstrate the applicability of the potentials, simulations on defect production by ion irradiation are carried out.

Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Glutamate mutase is comprised of two weakly associating subunits; E and S, that combine to form the coenzyme binding site. The active holoenzyme assembles in a kinetically complex process in which both the stoichiometry and apparent Kd for adenosylcobalamin (AdoCbl) are dependent upon the relative concentrations of the two subunits, as is the enzyme's specific activity. To facilitate mechanistic and structural studies on this enzyme we have genetically fused the S subunit to the C-terminus of the E subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment. This protein, GlmES, binds AdoCbl stoichiometrically and neither the affinity for AdoCbl nor the turnover number depends upon protein concentration. The kcat and Km for both substrate and coenzyme, together with the deuterium isotope effects on Vmax and Vmax/Km, have been determined for the GlmES-catalyzed reaction proceeding in both directions. Compared with wild type, the affinity for AdoCbl is unchanged, but for the conversion of L-glutamate to (2S,3S)-3-methylaspartate both kcat and Km for L-glutamate are decreased by about a third and the isotope effects are reduced, suggesting product release to be more rate-limiting. To test hypotheses concerning the activation of the coenzyme, we examined the binding of adenosylcobalamin, methylcobalamin, and cob(II)alamin to the enzyme. Each of these is bound with essentially the same affinity (2 microM), suggesting that, contrary to expectations, interactions between the protein and the adenosyl moiety do not serve to weaken the cobalt-carbon bond in the ground state.