Investigations presented here are (a) the study of reorientational dynamics and internal rotation in transition metal complexes by NMR relaxation experiments, and (b) the study of ligand exchange dynamics in transition metal complexes by exchange NMR experiments. The phenyl ring rotation in Ru3(CO)9(μ3-CO)(μ3-NPh) and Re(Co)2(CO)10(μ3- CPh) was monitored by 13C NMR relaxation experiments to probe intramolecular electronic and/or steric interactions. It was found that the rotation is relatively free in the first complex, but is restrained in the second one. The steric interactions in the complexes were ascertained by the measurement of the closest approach intramolecular distances. The rotational energy barriers in the two complexes were also calculated by using both the Extended Hiickel and Fenske-Hall methods. The study suggests that the barrier is due mainly to the steric interactions. The exchange NMR study revealed two carbonyl exchange processes in both Ru3(CO)9(μ3-CO)(μ3-NPh) and Ru3(CO)8(PPh3)(μ3-CO)(μ3-NPh). The lower energy process is a tripodal rotation of the terminal carbonyls. The higher energy process, resulting in the exchange between the equatorial and bridging carbonyls, but not between the axial and bridging carbonyls, involves the concerted formation of edge-bridging μ2-CO moieties. The effect of the PPh3 ligand on the carbonyl exchange rates has been discussed. A combination of relaxation and exchange NMR found that PPh3 ligand rotation about the Ru-P bond is slow on the exchange NMR time scale and the phenyl rotation about the P-Cipso bond is fast on the exchange NMR time scale but is slow on the NMR relaxation time scale.

Copper is an integral component of key metabolic enzymes. Numerous physiological processes depend on a fine balance between the biosynthetic incorporation of copper into proteins and the export of excess copper from the cell. The homeostatic control of copper requires the activity of the copper transporting ATPases (Cu-ATPases). In Wilson disease the disruption in the function of the Cu-ATPase ATP7B results in the accumulation of excess copper and a marked deficiency of copper-dependent enzymes. In this work, the structure of ATP7B has been modeled by homology using the Ca-ATPase X-ray structure, enabling a mechanism of copper transport by ATP7B to be proposed. The fourth transmembrane helix (TM4) of Ca-ATPase contains conserved residues critical to cation binding and is predicted to correspond to TM6 of the ATP7B homology model, containing the highly conserved CXXCPC motif. The interaction with Cu(I) and the importance of the 3 cysteines in TM6 of ATP7B has been shown using model peptides. ATP7B has a large cytoplasmic N-terminus comprised of six copper-binding domains (WCBD1-6), each capable of binding one Cu(I). Protein-protein interactions between WCBDs and the copper chaperone Atox1 has been shown, contrary to previous reports, to occur even in the absence of copper. 15N relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is minimal change in the dynamic properties and the relative orientation of the domains in the two states. The domain 4-5 linker remains flexible, and domain 5-6 is not a rigid dimer, with flexibility between the domains. Copper transfer to and between WCBD1-6 likely occurs via protein interactions facilitated by the flexibility of the domains with respect to each other. The flexible linkers connecting the domains are important in giving the domains motional freedom to interact with Atox1, to transfer copper to other domains, and finally to transfer copper to the transmembrane site for transport across the membrane.

Copper is an integral component of key metabolic enzymes. Numerous physiological processes depend on a fine balance between the biosynthetic incorporation of copper into proteins and the export of excess copper from the cell. The homeostatic control of copper requires the activity of the copper transporting ATPases (Cu-ATPases). In Wilson disease the disruption in the function of the Cu-ATPase ATP7B results in the accumulation of excess copper and a marked deficiency of copper-dependent enzymes. In this work, the structure of ATP7B has been modeled by homology using the Ca-ATPase X-ray structure, enabling a mechanism of copper transport by ATP7B to be proposed. The fourth transmembrane helix (TM4) of Ca-ATPase contains conserved residues critical to cation binding and is predicted to correspond to TM6 of the ATP7B homology model, containing the highly conserved CXXCPC motif. The interaction with Cu(I) and the importance of the 3 cysteines in TM6 of ATP7B has been shown using model peptides. ATP7B has a large cytoplasmic N-terminus comprised of six copper-binding domains (WCBD1-6), each capable of binding one Cu(I). Protein-protein interactions between WCBDs and the copper chaperone Atox1 has been shown, contrary to previous reports, to occur even in the absence of copper. 15N relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is minimal change in the dynamic properties and the relative orientation of the domains in the two states. The domain 4-5 linker remains flexible, and domain 5-6 is not a rigid dimer, with flexibility between the domains. Copper transfer to and between WCBD1-6 likely occurs via protein interactions facilitated by the flexibility of the domains with respect to each other. The flexible linkers connecting the domains are important in giving the domains motional freedom to interact with Atox1, to transfer copper to other domains, and finally to transfer copper to the transmembrane site for transport across the membrane.

The model-free analysis of NMR relaxation data, which is widely used for the study of protein dynamics, consists of the separation of the Brownian rotational diffusion from internal motions relative to the diffusion frame and the description of these internal motions by amplitude and timescale. Through parametric restriction and the addition of the Rex parameter a number of model-free models can be constructed. The model-free problem is often solved by initially estimating the diffusion tensor. The model-free models are then optimised and the best model is selected. Finally, the global model of all diffusion and model-free parameters is optimised. These steps are repeated until convergence. This thesis will investigate all aspects of the model-free data analysis chain. (For complete abstract open document)

Most theoretical treatments of Nuclear Magnetic Resonance (NMR) measurements of porous media assume ideal pore geometries for the pores (i.e. slabs, spheres or cylinders) with welldefined surface-to-volume ratios (S/V). This same assumption is commonly adopted for naturally occurring materials, where the pore geometry can differ substantially from these ideal shapes. In this paper the effect of the roughness of the pore surface on the T2 relaxation spectrum is studied. By homogenization of the problem using an electrostatic approach it is found that the effective surface relaxivity can increase dramatically in the presence of rough surfaces. This leads to a situation where the system responds as a pore with a smooth surface, but with significantly increased surface relaxivity. As a result the standard approach of assuming an idealized geometry with known surface to-volume and inverting the T2 relaxation spectrum to a pore size distribution is no longer valid. The effective relaxivity is found to be fairly insensitive to the shape of the roughness but strongly dependent on the width and depth of the surface geometry.

Relaxation in nuclear magnetic resonance is a powerful method for obtaining spatially resolved, timescale-specific dynamics information about molecular systems. However, dynamics in biomolecular systems are generally too complex to be fully characterized based on NMR data alone. This is a familiar problem, addressed by the Lipari-Szabo model-free analysis, a method that captures the full information content of NMR relaxation data in case all internal motion of a molecule in solution is sufficiently fast. We investigate model-free analysis, as well as several other approaches, and find that model-free, spectral density mapping, LeMaster’s approach, and our detector analysis form a class of analysis methods, for which behavior of the fitted parameters has a well-defined relationship to the distribution of correlation times of motion, independent of the specific form of that distribution. In a sense, they are all “model-free.” Of these methods, only detectors are generally applicable to solid-state NMR relaxation data. We further discuss how detectors may be used for comparison of experimental data to data extracted from molecular dynamics simulation, and how simulation may be used to extract details of the dynamics that are not accessible via NMR, where detector analysis can be used to connect those details to experiments. We expect that combined methodology can eventually provide enough insight into complex dynamics to provide highly accurate models of motion, thus lending deeper insight into the nature of biomolecular dynamics.

Nuclear magnetic resonance (NMR) provides a convenient probe for the study of molecular reorientation in liquids because nuclear spin-lattice relaxation times are dependent upon the details of molecular motion. The combined application of Raman and Infrared (IR) lineshape analysis can furnish more complete information to characterize the anisotropic rotation of molecules. Presented here are the studies of NMR relaxation times, together with Raman/IR Mneshape analysis of the solvent and temperature dependence of rotational diffusion in 1,3,5-tribromobenzene and 1,3,5-trifluorobenzene. In these experiments, it was found that the rotational diffusion constants calculated from Perrin's stick model were two to three times smaller than the measured values of D, and D,,. Similarly, rotational diffusion constants predicted by the Hu-Zwanzig slip model were too large by a factor of 2. Application of the newer Hynes-Kapral-Weinberg model furnished rotational diffusion constants that were in reasonable agreement with the experimental results. The vibrational peak frequencies and relaxation times of the isotropic Raman spectra of the υ1 modes of CD2Br2 and CHBr3 were studied in solution. The frequency shifts in non-interactive solvents were explained well on the basis of solution variations in the dispersion energy. In Lewis bases, the displacements were in some, but not all, cases greater than predicted. On the other hand, it was found that the vibrational relaxation times of the C-H/C-D modes decreased dramatically in all Lewis base solvents. Therefore, it was concluded that relaxation times of the υ1 modes, rather than frequency shifts, furnish a more reliable measure of hydrogen bonding interactions of halomethanes in solution.

Cette thèse concerne l’étude de phase de séparation de deux polymères thermosensibles connus-poly(N-isopropylacylamide) (PNIPAM) et poly(2-isopropyl-2-oxazoline) (PIPOZ). Parmi des études variées sur ces deux polymères, il y a encore deux parties de leurs propriétés thermiques inexplicites à être étudiées. Une partie concerne l’effet de consolvant de PNIPAM dans l’eau et un autre solvant hydromiscible. L’autre est l’effet de propriétés de groupes terminaux de chaînes sur la séparation de phase de PIPOZ. Pour ce faire, nous avons d’abord étudié l’effet de l’architecture de chaînes sur l’effet de cosolvant de PNIPAMs dans le mélange de méthanol/eau en utilisant un PNIPAM en étoile avec 4 branches et un PNIPAM cyclique comme modèles. Avec PNIPAM en étoile, l’adhérence de branches PNIPAM de à un cœur hydrophobique provoque une réduction de Tc (la température du point de turbidité) et une enthalpie plus faible de la transition de phase. En revanche, la Tc de PNIPAM en étoile dépend de la masse molaire de polymère. La coopérativité de déhydratation diminue pour PNIPAM en étoile et PNIPAM cyclique à cause de la limite topologique. Une étude sur l’influence de concentration en polymère sur l’effet de cosolvant de PNIPAM dans le mélange méthanol/eau a montré qu’une séparation de phase liquide-liquide macroscopique (MLLPS) a lieu pour une solution de PNIPAM dans le mélange méthanol/eau avec la fraction molaire de méthanol entre 0.127 et 0.421 et la concentration en PNIPAM est constante à 10 g.L-1. Après deux jours d’équilibration à température ambiante, la suspension turbide de PNIPAM dans le mélange méthanol/eau se sépare en deux phases dont une phase possède beaucoup plus de PNIPAM que l’autre. Un diagramme de phase qui montre la MLLPS pour le mélange PNIPAM/eau/méthanol a été établi à base de données expérimentales. La taille et la morphologie de gouttelettes dans la phase riche en polymère condensée dépendent de la fraction molaire de méthanol. Parce que la présence de méthanol influence la tension de surface des gouttelettes liquides, un équilibre lent de la séparation de phase pour PNIPAM/eau/méthanol système a été accéléré et une séparation de phase liquide-liquide macroscopique apparait. Afin d’étudier l’effet de groupes terminaux sur les propriétés de solution de PIPOZ, deux PIPOZs téléchéliques avec groupe perfluorodécanyle (FPIPOZ) ou groupe octadécyle (C18PIPOZ) comme extrémités de chaîne ont été synthétisés. Les valeurs de Tc des polymères téléchéliques ont beaucoup diminué par rapport à celle de PIPOZ. Des micelles stables se forment dans des solutions aqueuses de polymères téléchéliques. La micellization et la séparation de phase de ces polymères dans l’eau ont été étudiées. La séparation de phase de PIPOZs téléchéliques suit le mécanisme de MLLPS. Des différences en tailles de gouttelettes formées à l’intérieur de solutions de deux polymères ont été observées. Pour étudier profondément les différences dans le comportement d’association entre deux polymères téléchéliques, les intensités des signaux de polymères correspondants et les temps de relaxation T1, T2 ont été mesurés. Des valeurs de T2 de protons correspondants aux IPOZs sont plus hautes. / This thesis focused on the phase separation of two well-known thermoresponsive polymers, namely PNIPAM (poly(N-isopropylacrylamide)) and PIPOZ (poly(2-isopropyl-2-oxazoline). Despite various studies of the two polymers, two aspects of their thermal properties remained unclear and needed to be investigated. One is the cononsolvency effect of PNIPAM in water and a second water miscible solvent. The other is the effect of the end group properties on the phase separation of PIPOZ. With this in mind, we first studied the effect of the chain architecture on the cononsolvency of PNIPAM in water/methanol mixture, employing a 4-arm star shape PNIPAM and a cyclic PNIPAM as model. Tethering PNIPAM arms to a hydrophobic core resulted in a reduced Tc (cloud point temperature) and a lower phase transition enthalpy change. The Tc of the star shape PNIPAM was inversely dependent on the polymer molecular weight. The dehydration cooperativity was depressed for the star PNIPAM and cyclic PNIPAM due to topological constraints. A study of the effect of polymer concentration on the cononsolvency of PNIPAM in water/methanol mixture revealed a macroscopic liquid-liquid phase separation (MLLPS) for PNIPAM in water/methanol mixtures of methanol molar fraction ranging from 0.127 to 0.421 at a polymer concentration of 10 g·L-1. The turbid suspension of PNIPAM/water/methanol separated into a polymer rich phase coexisting with a polymer poor solution phase after equilibration for two days at room temperature. The phase diagram showing the MLLPS for the PNIPAM/water/methanol mixtures was constructed based on experimental data. The droplets in the condensed polymer rich phase showed a dependence on the methanol molar fraction. Methanol affects the surface tension of the liquid droplets. The slow equilibrium kinetics of PNIPAM phase separation was sped up and a macroscopic liquid-liquid phase separation realized. In order to study the effect of end groups on the solution properties of PIPOZ, two telechelic PIPOZ end capped with perfluorodecanyl groups (FPIPOZ) and octadecyl groups (C18PIPOZ), respectively, were synthesized. The Tc values of the telechelic polymers were greatly reduced after end-functionalization. Stable micelles formed in aqueous solutions of the telechelic polymers. The micellization and phase separation of the telechelic polymers in water were studied. The phase separation of the telechelic PIPOZs in water followed a liquid-liquid phase separation mechanism. Differences in the sizes of droplets formed inside of the two polymer solutions were observed. To further investigate the differences in the association behaviour between the two telechelic polymer, NMR signal intensities and T1 and T2 relaxation times were examined. Higher 1H T2 values were obtained for the IPOZ unit in FPIPOZ than that in C18PIPOZ, indicating a higher mobility of the main chain in the FPIPOZ micelles than that in the C18PIPOZ micelles. Together with the 13C NMR and 19F NMR relaxation studies, we obtained better knowledge of the association properties of the telechelic PIPOZ in water. NMR relaxation studies proved to be efficient way of probing the solution behaviour of the polymers.

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