Developing a triangular tessellation method for the analysis of medium ring pucker conformations

The main focus of this thesis is to investigate the relative conformational flexibilities of α-, β- and γ-cyclodextrins in water by analysing their macrocyclic ring puckering motion from Molecular Dynamics (MD) simulations. In particular, the puckering of the CDs is investigated through a coarse grained analysis of full atomistic simulations, where the CD conformational motions are studied on the macrocyclic scale rather than the atomistic scale. The flexibilities of the cyclodextrins (CDs) are then compared to their experimentally-observed aqueous solubility trend in order to try explain the anomalously flow solubility of β-cyclodextrin. β-CD has important applications in industry, such as the pharmaceutical industry, thus exploring the conformational reasons for its low solubility can help to design more effective cyclodextrin-based products in future. The ring puckering of the CDs is measured quantitatively using a reduced system of puckering coordinates based on the method of triangular tessellation. The triangular tessellation definition for monocylic 6-membered rings is first extended to 7- and 8-membered rings, and the corresponding puckering coordinates are derived mathematically. The macrocyclic CD rings are then simplified to monocyclic representations through an appropriate coarse graining of the molecules (specifically, α-, β- and γ-cyclodextrins are simplified to 6-, 7- and 8-sided rings, respectively), and the corresponding triangular tessellation definition is then used to measure their macrocyclic puckering. The rates of decay of the puckering motion are then calculated using time correlation functions, from which the relative flexibilities of the CDs is determined. Probability distributions are also used to investigate the ranges of the CD puckering. In addition, the horizontal contraction and expansion of the macrocyclic rings (termed """"breathing"""" herein) is analysed to supplement the puckering analysis. Puckering coordinates based on the triangular tessellation of 6-membered rings have been used previously to characterise all 38 canonical states of cyclohexane. In this thesis, a systematic procedure is developed to generate the triangular tessellation puckering coordinates of all the canonical states of 6-, 7- and 8-membered rings, and the coordinates for all canonical states of cycloheptane and cyclooctane are subsequently generated. These puckering coordinates can be useful not only in the conformational analysis of cyclohexane, cycloheptane and cyclooctane, but also to quantitatively characterise the conformations of 6-, 7- and 8-membered rings in general, both from experimental and computational studies

Developing methods to construct ring pucker free energy hypersurfaces applied to the analysis of glycosidase enzyme catalytic mechanisms

Includes bibliographical references.Carbohydrates consist of one or more sub-units usually various 5- and 6-membered cycles (furanoses and pyranoses) which can twist, bend or flip into a variety of conformers that differ in strain - this is ring puckering. These puckers notably the strained puckering conformers are observed during enzymatically assisted bond formation or cleavage of the glycosidic bonds of carbohydrate substrates. In this thesis, the free energy of ring puckering is calculated by implementing the Hill-Reilly reduced coordinate pucker description into the sampling enhancing Free Energies from Adaptive Reaction Coordinate Forces (FEARCF) method. FEARCF non-Boltzmann simulations of prototypical sugars β-Dribose and β-D-glucose converged to yield free energy pucker surfaces and volumes when using several semi-empirical QM methods - AM1, PM3, PM3CARB-1 and SCC-DFTB. From this, the accessible puckering conformations and minimum free energy paths of puckering were reasoned An analysis of the furanose and pyranose free energy pucker surfaces and volumes compared with both Density Functional Theory RB3LYP/6-311++G** optimised structures and a Hartree-Fock free energy surface revealed that SCC-DFTB provides the best semi-empirical description of 5- and 6- membered carbohydrate ring deformation. This illustrates that necessary high energy ring conformations observed in enzymatic binding sites requires the enzyme to induce and preserve high energy conformations required for successful hydrolyses and synthesis of the glycosidic bond. To further test this hypothesis, a 5- and 6-membered cycle were studied within enzymatic environments. The polysaccharide cellulose contains β 1-4 linked glucose subunit and is degraded by cellulase, a glycosidase. Specifically, the retaining cellobiohydrolase I (CBHI) of Trichoderma Reesei which cleaves cellobiose units from crystalline cellulose.The free energy volumes of puckering for the glucose sub-unit (in the catalytic position of an 8 unit cellulosic fragment - cellooctaose) were calculated and explored in vacuum, water and in the active site of CBHI. It was observed that the binding pocket of enzymes limits the ring pucker and that the active site amino acids preferentially stabilise certain puckering conformations. For CBHI, the first part of the glycosidase reaction is the glycosylation step. This was driven to completion during SCC-DFTB QM/MD FEARCF calculations where GLU212, ASP214 and GLU217 and part of the substrate were treated quantum mechanically. The general hybrid orbital method was used to connect the QM and MM regions. The free energy barriers of glycosylation were computed and the puckering statistics during the conversion of cellooctaose to products were correlated with this. Guanosine, a 5-membered ribose derivative is phosphorylated by Purine Nucleoside Phosphorylase (PNP) in order to salvage the guanine base. The effect of the PNP protein environment on ring pucker was studied by using FEARCF SCC-DFTB QM/MD non Boltzmann free energy calculations to quantify the pucker change induced in guanosine when changing environment from vacuum, to water and to the protein. In vacuo, the E4 and E1 pucker conformers were observed as minima. Upon solvation, the puckering phase space became less restricted with the 3T4 and 2T3 pucker conformers as minima. In the PNP active site pucker became restricted with only the 4E conformer observed

The development of hybrid quantum classical computational methods for carbohydrate and hypervalent phosphoric systems

Includes bibliographical references.Ab initio, density functional theory, and semi-empirical methods serve as major computational tools for quantum mechanical calculations of medium to large molecular systems. Semi-empirical methods are most effectively used in a hybrid quantum mechanics/molecular mechanics (QM/MM) dynamics framework. However, semi-empirical methods have been designed to provide accurate results for organic molecules, but often fail to treat hypervalent species accurately due to their use of an sp basis. Recently, significant breakthroughs have been made with the incorporation of d-orbitals into the semi-empirical framework, thereby allowing for accurate modeling of both hypervalent and transition metal systems. Here I consider two methods that adopt this new methodology, namely AM1/d-PhoT and AM1*. Our major focus is the simulation of chemical biological and more specifically chemical glycobiological problems of biochemical interest. When I tested the ability of both AM1/d-PhoT and AM1* to reproduce key metrics in chemical glycobiology (i.e., sugar ring pucker, phosphate participation in transferase reactions) these methods, in combination with the published parameters, performed very poorly. Using the AM1/d-PhoT and AM1* Hamiltonians I set out to re-parameterize these methods aiming to produce holistic biochemical QM/MM toolsets able to simulate fundamental problems of binding and enzyme reactivity in chemical glycobiology. We called these methods AM1/d-CB1 and AM1*-CB1. In the development of these parameter sets I focused specifically on proton transfer, carbohydrate ring puckering, bond polarization, amino acid interactions, and phosphate interactions (facets important to chemical glycobiology). Both AM1/d-CB1 and AM1*-CB1 make use of a variable property optimization parameter approach for the glycan molecular class and its chemical environment. The accuracy of these methods is evaluated for carbohydrates, amino acids and phosphates present in catalytic domains of glycoenzymes, and the are shown to be more accurate for key performance indices (puckering, etc.) and on average across all simulation derived properties (QM/MM polarization, protein performance, etc.) than all other NDDO semiempirical methods currently being used. A major objective of the newly developed AM1/d-CB1 and AM1*-CB1 is to provide a platform to accurately model reactions central to chemical glycobiology using hybrid QM/MM molecular dynamics (MD) simulations. AM1/d-CB1 is applied to a well-known reaction involving purine nucleoside phosphorylase (PNP) and results lead me to conclude that the method shows promise for modelling glycobiological QM/MM systems

POLYCYCLIC POLYAMINES: SYNTHESIS AND CONFORMATIONAL ANALYSIS

The synthesis, conformational analysis, and reactivity of a homologous series of tricyclic orthoamides is discussed. The tricyclic orthoformamides, orthoacetamides, orthopropionamides, and orthobenzamides were synthesized by the uncatalyzed condensation of macrocyclic triamines with amide acetals. The conformations were studied spectrally (IR, (\u271)H NMR, (\u2713)C NMR, DNMR) and by the application of empirical force field calculations (MM2). In most (but not all) cases the minimized conformations as generated by MM2 were found to be in agreement with the experimentally determined conformations. The alkylation, acylation, and hydrolysis of these compounds is also discussed. Efforts towards the synthesis of the spherically shaped host molecule 1,5,9,13,-tetraazatricyclo{7.7.3.3(\u275,13)}-docosane are described. A classical acylation-reduction sequence was employed in this synthesis. Cyclizations were carried out under high dilution conditions. The design and construction of a new high dilution apparatus is described. High yields of monomeric cyclic intermediates were obtained. Monomeric cyclic intermediates were purified by preparative gel permeation chromatography (GPC). The modification of a Waters 200 analytical GPC unit are described as are the column packing procedures for preparative GPC columns

A comparison of the Low Mode and Monte Carlo conformational search methods

The Low Mode (LM) and Monte Carlo (MC) conformational search methods were compared on three diverse molecular systems; (4R, 5S, 6S, 7R)-hexahydro-5,6-dihydroxy-1,3,4,7-tetrakis(phenylmethyl)-2H-1,3-diazapin-2-one (1), 2-methoxy-2-phenyl-2-triflouromethyl-N-α-methyl benzyl propanamide (2) and a trimeric 39-membered polyazamacrolide (3). We find that either method, or a combination of the methods, is equally efficient at searching the conformational space of the smaller molecular systems while a 50:50 hybrid of Low Mode and Monte Carlo is most efficient at searching the space of the larger molecular system

Triplex formation as monitored by EPR spectroscopy and molecular dynamics studies of spin -probe -labeled DNAs

Molecular modeling has proven to be a powerful tool for studying structure and dynamics of biologically important molecules. Since the advent of nitroxide based spin-probes the electron paramagnetic resonance (EPR) study of spin-labeled macromolecules has been able to provide insight into structural and dynamics properties of DNAs, proteins, and related systems. Spin labels have been extensively used to study the dynamics of oligonucleotides. An example of this is 5-membered ring nitroxide 5-(2,2,5,5-tetramethyl-3-ethynylpyrrolidine-1-oxyl)-uridine, which has been previously used in our laboratory to monitor triplex formation. Because of the difficult synthetic steps involved in the synthesis of this particular probe a new spin labeled DNA base, 5-(2,2,6,6-tetramethyl-4-ethynylpiperidyl-3-ene-1-oxyl)-uridine (6sp-uridine) is introduced in the current study. This spin label, 6sp, is readily prepared, in half the number of steps required for the previous one, and yet behaves in a spectroscopically analogous manner to its counterpart. The 6sp has been used here to detect the formation of a triplex DNA and to examine the relative rigidity of triplex DNA as compared to double stranded DNA using circular dichroism and EPR spectroscopy. Their EPR spectra show larger changes in response to differences in the mobility of the oligonucleotides they are attached to.;Extending their use in application to DNAs we have conducted Molecular Dynamics (MD) studies on six different oligonucleotides (ONs) molecules using the suite of programs contained in AMBER 5.0 with the Cornell force field. Quantum mechanical calculations at B3LYP level with standard 6-31G* basis set using Gaussian98 were performed. Together with available crystallographic data for different types of nitroxide molecules (Barone et al., J. Am. Chem. Soc. 1998, 120, 7069--7078), new parameters for NO· and sp-hybridized carbon moieties have been developed for the Cornell force field. MD simulations on single-stranded (ss), double-stranded (ds) and triple-stranded or triplex (tx) spin-probe labeled DNAs along with unmodified analogues have been studied over the course of 1 ns. Structural and conformational properties of DNA molecules are described from the analysis of the trajectories. Dynamics of the spin-label was characterized by correlation time (tau c). Our results indicate slower nitroxide motion associated with tx-DNA rather than ds- and ss-DNAs. The presence of spin labels has a substantial effect on the conformation of ss DNA, while ds- and tx-DNA is not affected by the introduction of labels. We have also shown that the presence of the spin-label has small stabilizing effect on ds and tx DNAs

Synthetic and Computational Studies on Polycyclic Aromatic Hydrocarbon Derivatives, Nucleoside Analogs and Peptides

In recent years, the understanding of the structure and functions of biological macromolecules has advanced rapidly, the result of which is a better mechanistic understanding of many biological processes. As an outgrowth of this understanding, organic molecules that react with biological macromolecules (DNA) or adopt conformations responsible for specific functions in biological macromolecules (peptides and proteins) have been synthesized and computational modeling studies performed. Polycyclic aromatic hydrocarbons (PAHs) and β-peptides are among synthetic organic compounds known to interact with natural biological macromolecules. This interaction may affect the specific biological functions of the biomacromolecules. A variety of synthetic methodologies have been employed in the synthesis of benzo[c]phenanthrene derivatives, single electron oxidation nucleoside adducts and deoxynucleoside derivatives (Part 1). In Part 2 heterogeneous backbone oligomers containing the β-amino acid, trans-2-aminocyclohexanecarboxylic acid (ACHC), and α-amino acids Ala, Phe, Val, Lys, and Tyr in an alternating sequence have been synthesized. Computational modeling studies have been applied in studying the diastereoselectivity of reaction intermediates in the PAH syntheses (Part 1), the interaction between the organic compounds and biomacromolecules (β-peptides with proteins Fos and Jun, Part 2), and the conformational preference (conformations of α/β-peptides, Part 2). Computational modeling based on molecular and quantum mechanical techniques were applied to complement the syntheses in Parts 1 and 2

Uptake mechanism of iron-phytosiderophore from the soil based on the structure of yellow stripe transporter

植物が根から鉄を吸収する機構の解明 --不良土壌を改善する次世代肥料の開発に期待--. 京都大学プレスリリース. 2022-12-08.Calcareous soils cover one-third of all land and cause severe growth defects in plants due to the poor water solubility of iron at high pH. Poaceae species use a unique chelation strategy, whereby plants secrete a high-affinity metal chelator, known as phytosiderophores (mugineic acids), and reabsorb the iron-phytosiderophore complex by the yellow stripe 1/yellow stripe 1-like (YS1/YSL) transporter for efficient uptake of iron from the soil. Here, we present three cryo-electron microscopy structures of barley YS1 (HvYS1) in the apo state, in complex with an iron-phytosiderophore complex, Fe(III)-deoxymugineic acid (Fe(III)–DMA), and in complex with the iron-bound synthetic DMA analog (Fe(III)–PDMA). The structures reveal a homodimeric assembly mediated through an anti-parallel β-sheet interaction with cholesterol hemisuccinate. Each protomer adopts an outward open conformation, and Fe(III)–DMA is bound near the extracellular space in the central cavity. Fe(III)–PDMA occupies the same binding site as Fe(III)–DMA, demonstrating that PDMA can function as a potent fertilizer in an essentially identical manner to DMA. Our results provide a structural framework for iron-phytosiderophore recognition and transport by YS1/YSL transporters, which will enable the rational design of new, high-potency fertilizers

COMPUTATIONAL STUDIES ON THE BINDING AND DYNAMICS OF THE OSH4 PROTEIN OF YEAST AND A MODEL YEAST MEMBRANE SYSTEM

Osh4 is an oxysterol binding protein homologue found in yeast that is essential for the intracellular transport of sterols. It has been proposed that Osh4 acts as a lipid transport protein, binding a single sterol residue and transporting it from the endoplasmic reticulum to the plasma membrane. The dynamics of Osh4 as well as ergosterol binding was observed using molecular dynamics simulations. Blind docking of several model lipid head group moieties was used to detect potential binding regions along the Osh4 surface favorable towards phospholipid interaction. Models frequently docked to a lysine-rich region on the side of the protein's β-barrel. A model ergosterol-containing membrane system for yeast was also constructed and simulated using molecular dynamics, and an improvement to the deuterium order parameters was observed over previous models. Understanding how Osh4 attaches to cellular membranes will lead to a clear understanding of how this protein transports sterols in vivo