Solid-state NMR on complex biomolecules: novel methods and applications

Abstract

Solid-state NMR (ssNMR) represents a versatile technique in providing atomic-resolution information without the need for crystals or fast molecular motion required for X-ray crystallography and solution-state NMR, respectively. Recent past has witnessed the ability of this technique in providing detailed structural information for many challenging biomolecules such as membrane proteins, amyloid fibrils,nucleoprotein complexes, and protein gels. However, other challenges still persist resulting in poor spectral resolution and sensitivity due to the presence of strong anisotropic interactions which are not averaged out by fast molecular motions as in the case of solution-state NMR. Hence further methodological advances are required to extend the use of ssNMR to study structure–function relationships in larger biomolecular systems. This thesis focuses on methodological aspectsand the application of solid-state NMR for the structural characterization of membrane proteins and amyloid fibrils. This thesis in the start discusses the application of fractional deuteration as a novel isotope-labeling scheme in ssNMR on a chimeric potassium channel KcsA-Kv1.3 embedded in lipid bilayers and its potential advantages in reducing spectral crowding, resolution enhancement in 1H MAS based ssNMR experiments and the establishment of structural constraints detecting long-range intra as well as intermolecular correlations in standard ssNMR correlation experiments. Secondly, a ssNMR-based hybrid strategy which employs structural constraints obtained from uniformly labeled [13C,15N] as well as fractionally deuterated [2H,13C,15N] versions of the chimeric potassium channel KcsA-Kv1.3 are used to construct 3D molecular structures. The approach is then used to characterize the channel before and after inactivation embedded in lipid bilayers. Furthermore, the structural stability as well as the influence of different lipid bilayer environment on the conformation and function of the potassium channel KcsA-Kv1.3 is probed using ssNMR spectroscopy. Later part of the thesis focuses on the structural characterization of amyloid forming proteins by ssNMR. Dedicated ssNMR methods are employed to investigate the overall fibril arrangements and to generate structural models of polyglutamine peptides that contain polyglutamine expansions of varying lengths for amyloid forming proteins, such as in the Huntingtin protein involved in Huntington’s disease. The last section of the thesis deals with the secondary structure characterization of a liposomal vaccine against Alzheimer’s disease using high resolution ssNMR spectroscopy in combination with other biophysical techniques. In particular, the secondary structure of a series of peptides used as antigen and their vaccine variants upon incorporation into liposomes has been investigated. Furthermore, the key parameter that control peptide conformation by modulation of the peptide lipidation pattern, spacer and liposome composition are studied.