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The topology and dynamics of biomolecules are intricately linked with their biological function. The focus of this thesis is the NMR-based measurement of topology and dynamics in biomolecular systems, and methods of measuring immersion depth and orientation of membrane-associated molecules. In detergent micelles and lipid bilayers, the local concentrations of hydrophobic and hydrophilic molecules are a function of their bilayer immersion depth. For paramagnetic molecular oxygen or metal cations, the magnitudes of the associated paramagnetic isotropic contact shifts and relaxation rate enhancements (PREs) are therefore depth-dependent. NMR measurements of these effects reveal the immersion depth of bilayer- or detergent-associated molecules. This work first explores transbilayer oxygen solubility and thermodynamics, as measured from contact shifts and PREs of the constituent lipid molecules in the presence of 30 bar oxygen. Contact shifts revealed the transmembrane O2 solubility profile spans a factor of seven across the bilayer, while PREs indicated that oxygen partitioning into bilayers and dodecylphosphocholine (DPC) micelles is entropically driven. Next, this work describes how paramagnetic effects from molecular oxygen and Ni(II) cations may be employed to study the immersion depth and topology of drug and protein molecules in DPC micelles. In one study, the positioning of the amphipathic drug imipramine in micelles was determined from O2- and Ni(II)-induced contact shifts. A second study, relying solely on O2-induced PREs, determined the tilt angles and micelle immersion depths of the two alpha helices in a monomeric mutant of the membrane protein phospholamban. A third study utilized 19F NMR to explore the importance of juxtamembraneous tryptophans on the topology of the membrane protein synaptobrevin, via O2-induced contact shifts and solvent-induced isotope shifts of a juxtamembraneous 19F-phenylalanine. Comparison of synaptobrevin constructs with zero, one, and two juxtamembraneous tryptophans revealed that while one tryptophan is sufficient to ‘anchor’ the protein in micelle, the addition of a second tryptophan dampens local dynamics. These solution state NMR studies demonstrate how paramagnetic effects from dissolved oxygen, complemented with measurements of local water exposure, provide detailed, accurate descriptions of membrane immersion depth and topology. These techniques are readily extended to the study of a wide range of biomolecules.

The topology and dynamics of biomolecules are intricately linked with their biological function. The focus of this thesis is the NMR-based measurement of topology and dynamics in biomolecular systems, and methods of measuring immersion depth and orientation of membrane-associated molecules. In detergent micelles and lipid bilayers, the local concentrations of hydrophobic and hydrophilic molecules are a function of their bilayer immersion depth. For paramagnetic molecular oxygen or metal cations, the magnitudes of the associated paramagnetic isotropic contact shifts and relaxation rate enhancements (PREs) are therefore depth-dependent. NMR measurements of these effects reveal the immersion depth of bilayer- or detergent-associated molecules. This work first explores transbilayer oxygen solubility and thermodynamics, as measured from contact shifts and PREs of the constituent lipid molecules in the presence of 30 bar oxygen. Contact shifts revealed the transmembrane O2 solubility profile spans a factor of seven across the bilayer, while PREs indicated that oxygen partitioning into bilayers and dodecylphosphocholine (DPC) micelles is entropically driven. Next, this work describes how paramagnetic effects from molecular oxygen and Ni(II) cations may be employed to study the immersion depth and topology of drug and protein molecules in DPC micelles. In one study, the positioning of the amphipathic drug imipramine in micelles was determined from O2- and Ni(II)-induced contact shifts. A second study, relying solely on O2-induced PREs, determined the tilt angles and micelle immersion depths of the two alpha helices in a monomeric mutant of the membrane protein phospholamban. A third study utilized 19F NMR to explore the importance of juxtamembraneous tryptophans on the topology of the membrane protein synaptobrevin, via O2-induced contact shifts and solvent-induced isotope shifts of a juxtamembraneous 19F-phenylalanine. Comparison of synaptobrevin constructs with zero, one, and two juxtamembraneous tryptophans revealed that while one tryptophan is sufficient to ‘anchor’ the protein in micelle, the addition of a second tryptophan dampens local dynamics. These solution state NMR studies demonstrate how paramagnetic effects from dissolved oxygen, complemented with measurements of local water exposure, provide detailed, accurate descriptions of membrane immersion depth and topology. These techniques are readily extended to the study of a wide range of biomolecules.

NMR spectroscopy of paramagnetic solids provides detailed information about the local configuration and the chemical environment of the NMR observed center, as well as about the structural, magnetic and electronic properties of the coordianted paramagnetic centres. In the case of complex paramagnetic solids such as cathode materials for (rechargeable) batteries, NMR represents an invaluable tool to provide insight into the structural and electronic properties of the systems, which are at the base of the electrochemical performance of these materials. However, the paramagnetism makes the interpretation of the NMR data very challenging. This is primarily due to the interactions of the unpaired electrons with the NMR observed nucleus, and the interpretation of the NMR spectra often requires the aid of reliable theoretical and computational methods. Often the dominant interaction contributing to the measured isotropic shifts is the hyperfine interaction between the unpaired electrons and the observed nucleus, which results from the transfer of unpaired electrons from the paramagnetic centre(s) to the NMR observed site. In systems such as the ones studied here, in which the paramagnetic ions are a major constituent of the lattice, the multitide of different local environments results in a complex distribution of resonances. As in the case of the Li$_x$V$_6$O$_{13}$ cathode material, a methodical investigation of the configurational stability from first principles gives insight into the preferred site configurations. The combination of experimental $^7$Li NMR spectra and hyperfine shift DFT calculations of the so-found stable Li environments allows to unravel the complex lithiation mechanism of this material. In the other case of the LiTi$_x$Mn$_{2-x}$O$_4$ cathode materials, the $^7$Li hyperfine shifts calculated from first principles for a variety of Li environments are combined in a lattice model which allows to assign the isotropic regions of the experimental $^7$Li NMR spectra, helping to resolve the complex cation ordering as a function of Mn/Ti content in the series. For paramagnetic centres with an unquenched orbital component of the electron magnetic moment(s), the spin-orbit coupling effects also contribute to the paramagnetic NMR shift and shift anisotropy. A first principles model is derived, which describes how spin-orbit coupling and the single-ion $g$-tensor are defined and calculated in periodic paramagnetic solids, and how they can be coupled with the hyperfine interaction to model their effects on the NMR spectrum. The method is applied to a series of olivine-type LiTMPO$_4$ cathode materials (with TM = Mn, Fe, Co, and Ni) and the respective $^7$Li and $^{31}$P NMR spectra are simulated and compared with the experiments. The other paramagnetic effect considered in this thesis involves the bulk magnetic susceptibility (BMS), which is particularly important for paramagnetic single crystals and solids of complex shape. The BMS effect results from the discontinuity of the bulk susceptibility at the surface of the crystal, inducing a demagnetizing field throughout the sample which changes the measured NMR shift and shift anisotropy. A method to analytically calculate the demagnetising field and the BMS shift in crystals of different shapes is derived, and it is applied to a series of LiFePO$_4$ single crystals for which the $^7$Li NMR spectra are also measured experimentally. The study confirms that, particularly for $^7$Li NMR, the macroscopic shape-dependent BMS shift can indeed be a significant contribution to the measured resonances, determining the large variation in shift measured for the crystals of different shapes.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23460号 / 理博第4754号 / 新制||理||1681(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 杤尾 豪人, 教授 森 和俊, 教授 望月 敦史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Understanding biological phenomena at atomic resolution is one of the keys to modern drug design. In particular, knowledge of 3D structures of proteins and their interactions with other macromolecules are necessary for designing chemical compounds that modify biological processes. Conventional methods for protein structure determinations comprise X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. These techniques can also determine the binding mode of chemical compounds. Either technique can be slow and costly, making it highly relevant to explore alternative strategies. Paramagnetic NMR spectroscopy is emerging as such an alternative technique. In order to measure the paramagnetic effects, two NMR spectra are compared that have been measured with and without a bound paramagnetic metal ion. In particular, pseudocontact shifts (PCS) of nuclear spins are easily measured as the difference (in ppm) of the chemical shifts between the two spectra. PCSs provide long range and orientation dependent restraints, allowing positioning of the spin with respect to the magnetic susceptibility tensor anisotropy (Δχ-tensor) of the metal ion. In this thesis, I used the PCS effect to computationally extract information from NMR spectra. I developed (i) a tool (called Possum) to automatically assign diamagnetic and paramagnetic spectra of the methyl groups of amino acid side chains, given structural information of the protein studied and prior knowledge of the Δχ-tensor; (ii) I designed a comprehensive software package (called Numbat) to extract Δχ-tensor parameters from assigned PCS values and the available 3D structure; and (iii) I incorporated PCS-based restraints into the protein structure prediction software CS-ROSETTA and demonstrated that this combination (PCS-ROSETTA) presents a significant improvement for de novo structure determination. The three projects serve different purposes at different stages of protein NMR studies. They could be combined in the following manner: Starting from assigned backbone PCSs, PCS-Rosetta could be used to determine the 3D structure of the protein. Possum can then be used to automatically assign the NMR resonances of the methyl groups using PCSs. Finally, Numbat can be used to fit improved Δχ-tensors to all the PCS data, analyze the quality of the Δχ-tensors and identify possible wrong assignments. Iterative repetition of this protocol would give a 3D structural model of the protein with a minimum of data. Alternatively, the Δχ-tensor parameters and PCSs could be used as input for a traditional software package such as Xplor-NIH to compute a 3D structure of the protein.

Understanding biological phenomena at atomic resolution is one of the keys to modern drug design. In particular, knowledge of 3D structures of proteins and their interactions with other macromolecules are necessary for designing chemical compounds that modify biological processes. Conventional methods for protein structure determinations comprise X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. These techniques can also determine the binding mode of chemical compounds. Either technique can be slow and costly, making it highly relevant to explore alternative strategies. Paramagnetic NMR spectroscopy is emerging as such an alternative technique. In order to measure the paramagnetic effects, two NMR spectra are compared that have been measured with and without a bound paramagnetic metal ion. In particular, pseudocontact shifts (PCS) of nuclear spins are easily measured as the difference (in ppm) of the chemical shifts between the two spectra. PCSs provide long range and orientation dependent restraints, allowing positioning of the spin with respect to the magnetic susceptibility tensor anisotropy (Δχ-tensor) of the metal ion. In this thesis, I used the PCS effect to computationally extract information from NMR spectra. I developed (i) a tool (called Possum) to automatically assign diamagnetic and paramagnetic spectra of the methyl groups of amino acid side chains, given structural information of the protein studied and prior knowledge of the Δχ-tensor; (ii) I designed a comprehensive software package (called Numbat) to extract Δχ-tensor parameters from assigned PCS values and the available 3D structure; and (iii) I incorporated PCS-based restraints into the protein structure prediction software CS-ROSETTA and demonstrated that this combination (PCS-ROSETTA) presents a significant improvement for de novo structure determination. The three projects serve different purposes at different stages of protein NMR studies. They could be combined in the following manner: Starting from assigned backbone PCSs, PCS-Rosetta could be used to determine the 3D structure of the protein. Possum can then be used to automatically assign the NMR resonances of the methyl groups using PCSs. Finally, Numbat can be used to fit improved Δχ-tensors to all the PCS data, analyze the quality of the Δχ-tensors and identify possible wrong assignments. Iterative repetition of this protocol would give a 3D structural model of the protein with a minimum of data. Alternatively, the Δχ-tensor parameters and PCSs could be used as input for a traditional software package such as Xplor-NIH to compute a 3D structure of the protein.

In der Kernspinresonanzspektroskopie (NMR) treten drei Effekte auf, die paramagnetische und diamagnetische Moleküle in isotroper Lösung unterscheiden: residuale dipolare Kopplung (RDC), Pseudokontaktverschiebung (PCS) und paramagnetische Relaxationsverstärkung (PRE). Alle drei Effekte sind abhängig von intermolekularen Winkeln und Abständen und können daher Informationen über die Struktur und Dynamik des Moleküls liefern. Um diese Informationen zu erhalten, muss das Molekül paramagnetische Eigenschaften aufweisen. Eine der heutzutage gebräuchlichen Methoden verwendet kleine molekulare Tags, die paramagnetische Metallionen koordinieren. Die meisten dieser Tags binden über eine Disulfidbrücke an Cysteine an der Proteinoberfläche. Um diese Methode für DNA anzuwenden werden daher neue Taggingstrategien benötigt. Im Rahmen dieser Arbeit wurde eine modifizierte Nukleobase synthetisiert, mit der ein Schwefelatom in die DNA eingebracht werden kann. Diese Methode erlaubt es, jeden Tag an die DNA zu binden, der als Verbindungsmethode eine Disulfidbrücke nutzt. Mit der Nukleobase wird eine Kohlenstoff-Dreifachbindung in die DNA eingefügt und mit Hilfe einer dipolaren Cycloaddition wird die freie Thiolgruppe eingebracht. Die modifizierte Nukleobase wurde erfolgreich an einem selbstkomplementären DNA-Strang (24 Nukleobasen) getestet. Die Nukleobase wurde während der Synthese der DNA eingefügt und der mit Lutetium, Terbium oder Thulium vorbeladene Cys-Ph-TAHA Tag wurde über eine Disulfidbrücke an die DNA gebunden. Die Beladung des Tags und die Taggingreaktion verliefen hierbei quantitativ. Nach diesem Erfolg war es ein Hauptaspekt dieser Arbeit, eine verlässliche und reproduzierbare Aufreinigungs- und Probenvorbereitungsmethode zu entwickeln. Diesem Punkt kommt besondere Bedeutung zu, da das Phosphatrückgrat der DNA, im Gegensatz zu Proteinen, Metallionen koordinieren kann. Im Theorieteil dieser Arbeit ist eine komplette Herleitung der drei Hauptmerkmale paramagnetischer NMR gegeben. Diese Herleitung beginnt bei Grundbegriffen des Magnetismus und neben den Gleichungen für RDCs, PCSs und PREs werden Ausdrücke für den dipolaren Hamiltonoperator, Kreuzrelaxationsraten, kreuzkorrelierte Relaxationsraten, durch Alignment induzierte RDCs, Korrelationsfunktionen und spektrale Dichten gegeben. Das zweite Thema dieser Arbeit basiert auf einem weiteren paramagnetischen Effekt. Um der reduzierten Empfindlichkeit der Kernspinresonanzspektroskopie verglichen mit anderen Spektroskopiemethoden entgegenzuwirken, wurden viele Methoden entwickelt, die auf eine Erhöhung der Polarisierung der Atomkerne zielen, d.h. um sogenannte hyperpolarisierte Kerne zu erzeugen. Eine dieser Methoden, die photochemisch erzeugte dynamische Kernpolarisierung (photo CIDNP), basiert auf kurzlebigen Radikalen, die durch direkte Laserbestrahlung der Probe im Magneten erzeugt werden. Im Rahmen dieser Arbeit wurde ein photo CIDNP Aufbau entworfen, gebaut und getestet. Die ersten Experimente und Resultate mit Triethylendiamin, L-Tyrosin und 3-Fluor-L-tyrosin zeigen die Vorteile und Grenzen dieser Methode auf. Für 3-Fluor-L-tyrosin wurde eine komplette Analyse des Relaxationsverhaltens, einschließlich der Kreuzrelaxation und der kreuzkorrelierten Relaxation, durchgeführt.

The functional oxides are performing materials showing interestant properties. The study of the local environment respect to the average struvture is essential for the deep understanding of the correlations between structure and properties ; this investigation of short and medium range can be performed with the use of solid state NMR techniques. In particular in this thesis three different classes of materials for applications in fields of optic and energy are considered : perovskite structure LaSrAlO4, the melilite system LaSr(Ga/Al)3O7 and the family of orthosylicates Li2(Fe/Mn)SiO4.