The primary aim of this thesis is to investigate and quantify the self-diffusion processes of gaseous molecules adsorbed in industrially relevant microporous zeolite materials using Pulsed Field Gradient Nuclear Magnetic Resonance (PFG NMR). The main body of this work involves the use of weakly adsorbing hydrocarbon gases (CH4, C2H6 and C3H¬8) adsorbed in a large pore β-zeolite structure. This thesis describes the development of a solely PFG NMR based technique for measuring the molecular displacements of these species at varying length-scales. This enabled the characterisation of self-diffusion regimes across zeolite beds and within individual zeolite crystallites. The characterisation of self-diffusion processes within single zeolite crystallites was critical with respect to accounting for quantitative discrepancies reported in the literature between PFG NMR and alternative measurement techniques. This approach also revealed that the transitions in the Gaussian probability distributions of the molecular displacements in the aforementioned self-diffusion regimes could be recorded by varying the experimental time-scale for observing molecular motion. This technique was extended to characterise the self-diffusion processes of the aforementioned hydrocarbons in small (≤ 1 μm) and large (≥ 15 μm) zeolite crystallites to investigate the dependence of this technique on zeolite geometry. It was found that the self-diffusion coefficients within single crystallites were in good agreement with one another, despite their differing crystallite geometries. This technique was subsequently used to study the self-diffusion behaviour of two-component hydrocarbon gaseous mixtures with differing sorption properties co-adsorbed in β-zeolite. Excellent chemical shift resolution was obtained for chemically similar species using NMR spectroscopy, relaxometry and diffusometry without the use of Magic Angle Spinning (MAS). This connoted that conventional PFG NMR is capable of precisely characterising individual species in real world multi-component systems. This thesis also describes the self-diffusion of ammonia in small pore chabazite structures, which are typically used in Selective Catalytic Reduction (SCR) processes. It was found that the self-diffusion coefficient of this strongly adsorbing species increased with molecular loading up to a certain point. This peculiar behaviour implied a strong concentration and inter-molecular dependence within the zeolite structure. Lastly, the techniques which were developed at high magnetic field strengths (300 MHz) were transferred to a lower field strength (43 MHz) benchtop spectrometer at the Johnson Matthey Technology Centre (JMTC). This describes the first characterisation of mass transport behaviour of weakly interacting sorbates in zeolites using a portable spectrometer. This presents an excellent opportunity for future off-line molecular displacement measurements to be made for complex and real-world systems in a matter of minutes.

The work presented in this thesis has focused on the development and implementation of two-dimensional (2D) nuclear magnetic resonance (NMR) correlation techniques to unambiguously discriminate and characterise competitive adsorption and mass transfer processes in porous materials. This has primarily involved investigations on porous oxides; in particular silicas and aluminas commonly used as catalyst supports. The techniques used are demonstrated to be capable of acquiring information relevant to the performance of heterogeneous catalysts and adsorbents across the hierarchy of length scales relevant to industrial processes. The methodologies associated with the acquisition and processing of 2D NMR correlation data were first established through the development of analytical models capable of simulating 2D signal attenuation data for T1-T2, D-T2 and T2-T2 experiments. Common artefacts were also discussed by means of experimental and simulated examples and, where appropriate, methods have been introduced for their prevention. The NMR relaxation behaviour of water saturating the pore space of silica was observed to correlate strongly with independent measurements of the activation energy of dehydroxylation, thus establishing NMR relaxometry as a tool for directly probing the surface energetics of silica surfaces. This interpretation of T1/T2 ratios differs from that in conventional applications of the technique which typically present the ratio as an indicator of surface-adsorbate interaction strength. Here, the T1/T2 ratios of three liquid probe molecules: ethanol, diethyl ether and cyclohexane, are used to investigate the influence of pore size and density of adsorption sites on relaxation behaviour. Competitive adsorption behaviour has been directly investigated through the acquisition of T1-T2 correlation data of binary liquid mixtures imbibed in various silica supports. These measurements, in combination with a newly developed model for the relaxation of multi-component mixtures, have provided a comprehensive assessment of the ability of this technique to quantify intra-pellet compositions and address competitive adsorption behaviour in porous media. With the aid of a random walk Monte-Carlo model to simulate transverse relaxation in packed bed reactors, T2-T2 relaxation exchange measurements have been used to investigate mass transfer across the fluid-solid boundary in a packed bed reactor filled with γ-alumina and liquid cyclohexane. These data were used to quantify the rate of exchange between intra- and inter-pellet environments at a number of flow rates. Exchange rates were then converted into the more convenient terms of mass transfer coefficients and compared against literature data using two separate dimensionless mass transfer analyses.

abstract: Structural details of phosphonic acid functionalized nanomaterials and protic ionic liquids (PILs) were characterized using nuclear magnetic resonance (NMR) spectroscopy. It is well known that ligands play a critical role in the synthesis and properties of nanomaterials. Therefore, elucidating the details of ligand-surface and ligand-ligand interactions is crucial to understanding nanomaterial systems more completely. In an effort to further the understanding of ligand-surface interactions, a combination of multi-nuclear (1H, 29Si, 31P) and multi-dimensional solid-state NMR techniques were utilized to characterize the phosphonic acid functionalization of fumed silica nanoparticles using methyl phosphonic acid (MPA) and phenyl phosphonic acid (PPA). Quantitative 31P MAS solid-state NMR measurements indicate that ligands favor a monodentate binding mode. Furthermore, 1H-1H single quantum-double quantum (SQ-DQ) back-to-back (BABA) 2D NMR spectra of silica functionalized with MPA and PPA indicate that the MPA and PPA are within 4.2±0.2 Å on the surface of the nanomaterial. The ligand capping of phosphonic acid (PA) functionalized CdSe/ZnS core-shell quantum dots (QDs) was investigated with a combination of ligand exchange, solution and solid-state 31P NMR spectroscopy. In order to quantify the ligand populations on the surface of the QDs, ligand exchange facilitated by PPA resulted in the displacement of the PAs, and allowed for quantification of the free ligands using 31P liquid state NMR. In addition to characterizing nanomaterials, the ionicity and transport properties of a series of diethylmethylamine (DEMA) based protic ionic liquids (PILs) were characterized, principally utilizing NMR. Gas phase proton affinity was shown to be a better predictor for the extent of proton transfer, and in turn the ionicity of the PIL, than using ∆pKa. Furthermore, pulsed field gradient (PFG) NMR was used to determine that the exchangeable proton diffuses with the cation or the anion based on the strength of the acid used to generate the PILs. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015

Neste trabalho, apresentamos um estudo por Ressonância Magnética Nuclear (RMN), Análises Termogravimétricas (DSC e TG) e Espectroscopia Infravermelho em cornplexos poliméricos formados entre poli(óxido de etileno), POE, e sais de lítio. Estes complexos tem mostrado uma grande potencialidade em aplicações tecnológicas (baterias, sensores, etc). Desenvolvemos e caracterizamos sensores de umidade e, discutimos como a umidade afeta a conformação do complexo e a mobilidade das espécies iônicas e da cadeia polimérica. Nossos resultados indicam que a hidratação afeta a conformação dos complexos poliméricos através do efeito plasticizante da água, o qual induz uma expansão volumétrica na cadeia do POE. Entretanto, para os níveis de hidratação utilizados, o processo mostrou-se completamente reversível. Através do estudo de RMN, conseguimos diferenciar os movimentos da cadeia polimérica daqueles das espécies iônicas (cátion e ânion). A análise do segundo momento das formas de linha de ressonância e os estudos de relaxação nuclear nos permitiu estimar distâncias médias entre as espécies iônicas e os prótons da cadela nestes complexos. Observamos também, que o comportamento da relaxação spin rede do hidrogênio e do flúor no P(OE)-LiBF4, em função da temperatura e da freqüência, reflete a natureza desordenada do material e a complexidade do processo de condução iônica nestes sistemas / In this work we report on a study using Nuclear Magnetic Resonance (NMR), Thermogravimetry Analysis (TGA), Differential Scanning Calorimetric (DSC) and Infrared Spectroscopy in polymeric complexes formed between poly(ethylene oxide), PEO), and lithium salts. These complexes have shown a large potential in for technological applications in batteries, sensors, etc. We developed and characterized humidity sensors and we discussed how the humidity affects the conformation of the complexes, the mobility of ionic species, and the polymeric chains. The results indicate that the hydration effects the conformation of polymeric complexes by plasticizing the water, which induces a volumetric expansion in the PEO chain. The process was completely reversible for the level of hydration studied. NMR was used to distinguish the movement of polymeric chains from the movement of the ionic species. From the analysis of the second moment of resonance lines and from the study of the nuclear relaxation we were able to estimate the average distance between the ionic species and the proton In the complexes chains. The behavior of spin-lattice relaxation of hydrogen and flurine in the P(EO) LiBF4 as a function of temperature and frequency reflects the nature of the disorder and the complexity of the ionic conduction process In these materials

Discrete and Continuous spatial encoding methods are described with details of understanding principles and practical implications. Step by step experimental op- timization procedure of these methods to achieve slice selection are also discussed. In the subsequent chapters we use these methods for different applications. Spin-lattice relaxation parameters of NMR active nuclei provide valuable infor- mation on molecular dynamics. Single scan selective excitation methods of mea- surement of T1 result in significant reduction of time compared to the standard inversion recovery method and are attractive tools of applications in `Real time' NMR investigations of biological and chemical processes. It is shown here that the addition of the gradient echo following the selective excitation not only significantly improves the S/N ratio, but also makes GESSIR a versatile pulse sequence. Using this sequence, T1 values ranging from 2 s to 56 s have been measured with accuracy comparable to the standard IR experiment. This indicates that it is possible to utilize GESSIR for a wide range of molecules containing protons and hetero nuclei with medium to long T1 relaxation times as a routine NMR technique. The utility of the technique for studying other relaxation parameters has also been demonstrated. It may be mentioned that for measurement of relaxation parameters routinely, a few well-chosen points are enough. A fine selection of large number of experimental points could be useful when high accuracy is required or Chapter 3. GESSIR 91 for applications where certain property of the system investigated is changing in a continuous manner spatially and requires large number of slices to be selected as discussed in the next chapter. The long duration of time-honored two dimensional experiments is reduced to fraction of seconds by employing the ultrafast encoding experiments. Main com- plications in making the UF experiments available for routine use were the limited spectral widths and resolution in both UF and conventional dimensions. Various developments have been made in the path of improvements in increasing the spectral width in UF dimension. Of these, two experimental methods that are already proposed, namely the folding of peaks into the observable spectral window and the interleaved acquisition which doubles the spectral widths in both dimensions. The integration of covariance processing with ultrafast technique yields better correlated spectrum with considerable improvement in resolution. The effectiveness of the new processing is demonstrated for UF COSY experiments which can be easily extended to other ultrafast homonuclear experiments like TOCSY, NOESY as well as multi dimensions. The proposed processing method is an initial step to work on improving resolutions of UF data and making the ease of applicability of ultrafast spectroscopy as a routine multidimensional NMR. At the same time of this work W. Qui et.al [268] proposed a processing method based on covariance and pattern recognition for improving resolutions of spatially encoded data. They used pattern recognition algorithm also for avoiding the artifacts due to very low resolution data available with the UF experiment. They implemented the method UF TOCSY spectra and shown resolution improvement with the covariance pro- cessing for relatively more number of detection gradients which is many times hardware limited. Our method of covariance data processing is essentially same as that of Qui, less number of acquisition gradients were used in our processing, linear prediction and apodization concepts were utilized and the artifacts arise due mismatch of datas with positive and negative acquisition gradients are minimized by shifting one the data. In conclusion new methods of processing/the combination of various processing methods of the ultrafast data have the scope of improving the quality of ultrafast NMR spectra.

Solid-state Nuclear Magnetic Resonance (NMR)is one of the premiere biophysical methods that can be applied for addressing the structure and dynamics of biomolecules, including proteins, lipids, and nucleic acids. It illustrates the general problem of determining the average biomolecular structure, including the motional mean-square amplitudes and rates of the fluctuations. Lineshape and relaxtion studies give us a view into the molecular properties under different environments. To help the understanding of NMR theory, both lineshape and relaxation experiments are conducted with hexamethylbezene (HMB). This chemical compound with a simple structure serves as a perfect test molecule. Because of its highly symmetric structure, its motions are not very difficult to understand. The results for HMB set benchmarks for other more complicated systems like membrane proteins. After accumulating a large data set on HMB, we also proceed to develop a completely new method of data analysis, which yields the spectral densities in a body-fixed frame revealing internal motions of the system. Among the possible applications of solid-state NMR spectroscopy, we study the light activation mechanism of visual rhodopsin in lipid membranes. As a prototype of G-protein-coupled receptors, which are a large class of membrane proteins, the cofactor isomerization is triggered by photon absorption, and the local structural change is then propagated to a large-scale conformational change of the protein. Facilitation of the binding of transducin then passes along the visual signal to downstream effector proteins like transducin. To study this process, we introduce 2H labels into the rhodopsin chromophore retinal and the C-terminal peptide of transducin to probe the local structure and dynamics of these two hotspots of the rhodopsin activation process. In addition to the examination of local sites with solid-state 2H NMR spectroscopy, wide angle X-ray scattering (WAXS) provides us the chance of looking at the overall conformational changes through difference scattering profiles. Although the resolution of this method is not as high as NMR spectroscopy, which gives information on atomic scale, the early activation probing is possible because of the short duration of the optical pump and X-ray probe lasers. We can thus visualize the energy dissipation process by observing and comparing the difference scattering profiles at different times after the light activation moments.

Escherichia coli MutS is a DNA binding repair protein (97 kDa, monomer) and its biological significance arises from its recognition of mismatches which occur as errors during DNA replication. Mismatches and mutagenic bases represent a fascinating and diverse range of shapes and sizes and it is not obvious how a single protein (MutS) can recognise such molecular diversity against a huge background of canonical Watson-Crick base pairs. In this project, the structure of a 17mer mismatch GT DNA was carried out using NMR spectroscopy to identify the differences caused by the introduction of a non-canonical base pair on helical structure. The resulting structure was B-form in conformation and local helical distortions were observed about the GT mispair due primarily to its sheared orientation. The effect of mismatch orientation, sequence context and oligonucleotide length on mismatch stability was also investigated using UV absorbance melting and NMR spectroscopy. The results showed that substitution of a TG mispair for a GT mispair was accompanied by a small drop in melting temperature. It was also discovered that sequences in which purine-purine or pyrimidine-pyrimidine stacking occurred induced additional stability of the mismatch resulting in a higher melting temperature of the duplex.Affinity of mismatch GT DNA and its mismatch orientation, sequence context and length analogues for MutS was investigated by monitoring changes to the chemical shifts and linewidths of imino protons resonances during NMR titration. The results showed that MutS displayed higher affinity towards sequences which involved better stacking between the flanking base pairs and the GT/TG mispair.Analogous NMR structural investigations of 6-thioguanine modified 13mer GC DNA and its oxidised derivatives have been successfully carried out. The NMR structure was successfully determined of the former and the results obtained showed the effect on helical structure induced by the substitution of a different DNA lesion.Although the crystal structures of MutS bound to DNA mismatches have been known for a number of years, the analogous crystal structures of uncomplexed apo MutS have not been determined to date. Consequently, vital structural knowledge on the large change in conformation of MutS upon binding to the DNA mismatch is seriously lacking. We have successfully isolated the structurally and functionally important NTD of E. coli MutS and its labelled (13C, 15N) analogues and have shown that it is endowed with a stable, tertiary structural fold and well suited to NMR structure determination. This is exemplified by the assignments of several backbone amide and side chain resonances using isotope aided 3D NMR techniques.

Type IV pili, or fimbriae, are long, thin proteinaceous appendages found on the surface of many well-known pathogens. They mediate a variety of important virulence functions for the organism, such as twitching motility, biofilm formation, uptake of genetic material and host cell recognition and adhesion. Pili are formed by the rapid polymerization and de-polymerization of the pilin subunit, and this is orchestrated by a complex macromolecular machine which spans the bacterial cell envelope, requiring a variety of gene products. The type IV pilus biogenesis system is closely related to the bacterial type II secretion system, one of six designated multi-protein cell envelope complexes which are dedicated to the specific secretion of exotoxins and virulence factors. Many of these secretion systems also produce fimbrial structures to facilitate the extrusion of their substrates or to communicate with the host. As they form crucial virulence factors, the secretion systems and the type IV pilus biogenesis system have become attractive potential antimicrobial targets and obtaining structural and functional information for the components of these systems is an important first step towards achieving this.Type IV pili appear on the surface of bacteria through an outer membrane pore, PilQ, which is a member of the secretin family. Secretins are also found in the type II and III secretion systems, but the way in which they are regulated remains unclear. PilQ forms a dodecameric chamber in the outer membrane with a large vestibule which reaches into the periplasm, composed of its N-terminal domains. In this project, N-terminal domains from PilQ were produced in recombinant form and their structures determined by NMR. One of these domains revealed an eight-stranded beta-sandwich structure which appears to be unique to type IV pilus secretins and has not been structurally characterized before. Another revealed an alpha/beta type fold which is common to secretins of other systems. In the second part of this project, the interaction formed between the N-terminal alpha/beta domains of PilQ and an essential inner membrane-anchored lipoprotein, PilP, was probed by NMR chemical shift perturbation. Based on changes to the 15N-HSQC spectra the binding site was mapped onto each protein to produce a computational model for the complex formed between the two. Using a recent cryo-EM structure for the Neisseria PilQ dodecamer determined by colleagues, it was possible to model the PilQ N-terminal domains in complex with PilP into the electron density map. This produced a model for the trans-periplasmic assembly formed by PilQ and PilP in the type IV pilus biogenesis system, and led to the conclusion that the PilQ dodecamer needs to disassemble considerably at the base to accommodate a pilus fibre. The novel beta-domains might therefore function to gate or open the secretin, and PilP may play a role in stabilizing the secretin during this and serve to connect the outer and inner membrane system components.

Magister Scientiae - MSc / Endozepine is an alternative name for the testis-specific isoform of the acyl-CoA binding protein (t-ACBP). Acyl-CoA binding proteins form a highly conserved family of proteins, which bind long chain fatty acid esters with nanomolar affinity. They are also known to be endogenous ligands to the --amino butyric acid (GABA) receptor in the central nervous system and to play a role in a wide variety of cellular functions such as vesicular trafficking, fatty acid biosynthesis and gene regulation. A role for endozepine in apoptosis was suggested through promoter gene trapping studies using CHO22 cells in which 90 % reduction in the expression of endozepine correlated with delayed mitochondrial permeabilization, a reduced activation of caspase-3 (an activator of apoptosis) and a consequent resistance to C2-ceramide induced apoptosis. Transduction studies using Tat-GFP-ELP fusion protein showed that endozepine restored the sensitivity of mutant CHO22 cells to C2-ceramide induced apoptosis. In this thesis, we have investigated two hypotheses for the involvement of endozepine in ceramide-induced apoptosis. The first hypothesis is that endozepine contributes to apoptosis through the transport of palmitoyl-CoA, a substrate required for the de novo synthesis of ceramide. The second hypothesis is that endozepine interacts directly with ceramide leading to interaction with peripheral benzodiazepine receptor and a subsequent opening of the mitochondria permeability transition pore, leading to apoptosis. / South Africa

Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN- complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.