Aggregate Polymorphism in Protein Deposition Diseases: Investigations by Magic Angle Spinning Solid State NMR and Transmission Electron Microscopy

The deposition of normally soluble protein can occur in any organ in the human body and is associated with tissue dysfunction, cell death, and the progression of disease. Protein aggregation is concomitant with blindness as an outcome of cataract, life-threatening organ failure as a consequence of amyloidosis, and pronounced degeneration of the brain. The mutation responsible for Huntington’s disease causes an expansion of the polyglutamine domain of huntingtin exon 1 that directly promotes misfolding and refolding of huntingtin and huntingtin N-terminal fragments into amyloid-like fibrils in the basal striatum and cortex of the brain. Several fibril polymorphs have been identified, however the relationship between neurotoxicity and amyloid polymorphism is poorly understood. The P23T mutant of gamma-D-crystallin is associated with cataract formation in the eyes of very young children. Crystallins have been shown to form amyloid-like, native-like, as well as amorphous looking aggregates in vitro, accordingly it is unclear which class of aggregates P23T gamma-D-crystallin is most likely to form in cataract. Apolipoprotein A-I is a known anti-atherosclerotic factor and oxidation at methionine residues enhances its function. However, this oxidation also induces aggregation in vascular amyloidosis, which is interlinked with atherosclerosis progression. It is unclear whether apolipoprotein A-I aggregates misfold into amyloid-like fibrils as is usually the case in amyloidosis. Magic angle spinning solid state NMR (MAS ssNMR) is ideally suited to provide atomic resolution information on the structure and dynamics of insoluble, non-crystalline protein aggregates. Transmission electron microscopy (TEM) allows for the visualization of morphological features of aggregates that cannot be observed by optical microscopy and can be used to identify polymorphs and aid in distinguishing between different classes of aggregates. In this dissertation, I use both MAS ssNMR and TEM in addition to other biophysical and structural techniques to investigate the differences in structure and dynamics between polymorphs of huntingtin exon 1, P23T gamma-D-crystallin, and apolipoprotein A-I. Enabled by my experiments, I narrow down the potential molecular mechanisms involved in these three distinct types of protein deposition diseases. I show that depending on the milieu, proteins have the potential for varied amyloidogenic and non-amyloidogenic self-assembly

Triazolium-Based Energetic Ionic Liquids

The energetic ionic liquids formed by the 1,2,4-triazolium cation family and dinitramide anion are investigated by ab initio quantum chemistry calculations, to address the following questions:  How does substitution at the triazolium ring\u27s nitrogen atoms affect its heat of formation, and its charge delocalization? What kind of ion dimer structures might exist? And, do deprotonation reactions occur, as a possible first step in the decomposition of these materials

On the use of ultracentrifugal devices for routine sample preparation in biomolecular magic-angle-spinning NMR

A number of recent advances in the field of magic-angle-spinning (MAS) solid-state NMR have enabled its application to a range of biological systems of ever increasing complexity. To retain biological relevance, these samples are increasingly studied in a hydrated state. At the same time, experimental feasibility requires the sample preparation process to attain a high sample concentration within the final MAS rotor. We discuss these considerations, and how they have led to a number of different approaches to MAS NMR sample preparation. We describe our experience of how custom-made (or commercially available) ultracentrifugal devices can facilitate a simple, fast and reliable sample preparation process. A number of groups have since adopted such tools, in some cases to prepare samples for sedimentation-style MAS NMR experiments. Here we argue for a more widespread adoption of their use for routine MAS NMR sample preparation

Atomic spectral-product representations of molecular electronic structure: metric matrices and atomic-product composition of molecular eigenfunctions

Recent progress is reported in development of ab initio computational methods for the electronic structures of molecules employing the many-electron eigenstates of constituent atoms in spectral-product forms. The approach provides a universal atomic-product description of the electronic structure of matter as an alternative to more commonly employed valence-bond- or molecular-orbital-based representations. The Hamiltonian matrix in this representation is seen to comprise a sum over atomic energies and a pairwise sum over Coulombic interaction terms that depend only on the separations of the individual atomic pairs. Overall electron antisymmetry can be enforced by unitary transformation when appropriate, rather than as a possibly encumbering or unnecessary global constraint. The matrix representative of the antisymmetrizer in the spectral-product basis, which is equivalent to the metric matrix of the corresponding explicitly antisymmetric basis, provides the required transformation to antisymmetric or linearly independent states after Hamiltonian evaluation. Particular attention is focused in the present report on properties of the metric matrix and on the atomic-product compositions of molecular eigenstates as described in the spectral-product representations. Illustrative calculations are reported for simple but prototypically important diatomic (H_2, CH) and triatomic (H_3, CH_2) molecules employing algorithms and computer codes devised recently for this purpose. This particular implementation of the approach combines Slater-orbital-based one- and two-electron integral evaluations, valence-bond constructions of standard tableau functions and matrices, and transformations to atomic eigenstate-product representations. The calculated metric matrices and corresponding potential energy surfaces obtained in this way elucidate a number of aspects of the spectral-product development, including the nature of closure in the representation, the general redundancy or linear dependence of its explicitly antisymmetrized form, the convergence of the apparently disparate atomic-product and explicitly antisymmetrized atomic-product forms to a common invariant subspace, and the nature of a chemical bonding descriptor provided by the atomic-product compositions of molecular eigenstates. Concluding remarks indicate additional studies in progress and the prognosis for performing atomic spectral-product calculations more generally and efficiently

Cataract-associated P23T γ 3D-crystallin retains a native-like fold in amorphous-looking aggregates formed at physiological pH

Cataracts cause vision loss through the large-scale aggregation of eye lens proteins as a result of ageing or congenital mutations. The development of new treatments is hindered by uncertainty about the nature of the aggregates and their mechanism of formation. We describe the structure and morphology of aggregates formed by the P23T human Î 3D-crystallin mutant associated with congenital cataracts. At physiological pH, the protein forms aggregates that look amorphous and disordered by electron microscopy, reminiscent of the reported formation of amorphous deposits by other crystallin mutants. Surprisingly, solid-state NMR reveals that these amorphous deposits have a high degree of structural homogeneity at the atomic level and that the aggregated protein retains a native-like conformation, with no evidence for large-scale misfolding. Non-physiological destabilizing conditions used in many in vitro aggregation studies are shown to yield qualitatively different, highly misfolded amyloid-like fibrils

Pentazole-Based Energetic Ionic Liquids: A Computational Study

The structures of protonated pentazole cations (RN5H+), oxygen-containing anions such as N(NO2)2-, NO3-, and ClO4- and the corresponding ion pairs are investigated by ab initio quantum chemistry calculations. The stability of the pentazole cation is explored by examining the decomposition pathways of several monosubstituted cations (RN5H+) to yield N2 and the corresponding azidinium cation. The heats of formation of these cations, which are based on isodesmic (bond-type conserving) reactions, are calculated. The proton-transfer reaction from the cation to the anion is investigated