High-resolution solid-state NMR (ssNMR) has recently emerged as a powerful characterization technique for systems that cannot be investigated by solution NMR or X-ray crystallographic methods, and represents a subtle complementary technique for any atomic-scaled study. This is particularly true in structural biology. There exist nowadays well established protocols for sample preparation, resonance assignment and collection of structural restraints, that have paved the way to the first three-dimensional structure determinations at atomic resolution of biomolecules in the solid state, from microcrystalline samples to fibrils and membrane-associated systems. Despite rapid uptake in the field of structural biology, however, these methods for structure determination are far from being routine, and several important problems remain however to be solved before ssNMR is applied to the study of challenging solid protein assemblies. Many methodological developments are still expected in this fast evolving field. Most of the model systems used up-to-date for method development in biological solid-state NMR, are relatively small globular proteins, in the range of 50 to 80 residues (approximately 5.5 to 9.5 kDa). In order to extend the capabilities of ssNMR to larger substrates, the objectives of this thesis are twofold: a) to establish a new, large and more complex model system, and b) to develop new, sophisticated NMR experiments in order to improve the sensitivity and the resolution of the currently existing schemes for resonance assignment, which is one of the main barrier to progress to structural investigation in solid proteins. The N-terminal domain of the subunit of E. coli DNA polymerase III (186 : 186 residues, 18 kDa) was selected as a target. This domain represents the catalytic core of the E. coli replisome, the large molecular machine that replicated DNA in bacteria. In a first part, preparation conditions for solid-state NMR are obtained, notably in combination with automated screening processes for high-throughput protein crystallography, and almost complete resonance assignment is performed by the application of established experiments based on high-power rf irradiations and slow magic-angle spinning (MAS) at high magnetic fields. In a second part, we explore the use of MAS at so-called ultra-fast spinning rates (60 kHz). We show that this makes possible the use of “totally low power” experiments. This yields an extraordinary increase in resolution and sensitivity, enabling the acquisition of selective cross- polarization (CP) transfers, through-bond correlations and 1 H-detected correlations. In particular, we demonstrate that narrow 1 H NMR line widths can be obtained for fully protonated protein samples in the solid state under ultra-fast magic-angle spinning for medium-size microcrystalline and non-crystalline proteins, without any need for dilution against a deuterated background. This provides extensive, robust and expeditious assignments of the backbone 1 H, 15 N, 13 Cα and 13 CO resonances of proteins in different aggregation states, without the need of deuteration. The final part of this thesis concerns the study of thermotropic liquid crystals (LC or LX) phases of a de Vries smectogen, the (S)-hexyl-lactate derivative abbreviated as 9HL, selectively deuterated in a phenyl moiety of the aromatic core. de Vries mesophases show a substantially constant layer spacing in the transition between smectic C and smectic A mesophases and are for this reason of great interest for the development of new ferroelectric (FLC) and antiferroelectric (AFLC) electrooptic devices. Our work is the first attempt to apply NMR to characterize the nature of the de Vries transition, discriminating among possible models. It is also one of the first examples in the scientific literature of application of high magnetic field (above 16 T) for the analysis of LX phases.
Sviluppi metodologici per la cristallizzazione e l’analisi strutturale di proteine tramite Risonanza Magnetica Nucleare allo stato solido / Marchetti, Alessandro. - (2012 Jul 12).
|Titolo:||Sviluppi metodologici per la cristallizzazione e l’analisi strutturale di proteine tramite Risonanza Magnetica Nucleare allo stato solido|
|Altri titoli:||D eveloppements m ethodologiques pour la cristallisation et l'analyse structurale de prot eines par R esonance Magn etique Nucl eaire en phase solide|
|Relatore/i esterno/i:||Emsley, Lyndon|
|Data di pubblicazione:||2012-07-12|
|Parole chiave (inglese):||Chemistry|
de Vries smectogens
DNA polymerase III
high-throughput protein crystallization techniques
|Editore:||Scuola Normale Superiore|
|Appare nelle tipologie:||9.1 Tesi di Dottorato|