Deo, Thomas Sandeep
(2021)
Applications of Dynamic nuclear polarisation in biological systems.
PhD thesis, University of Nottingham.
Abstract
Cellular membrane disruption induced by the aggregation of amyloid beta (Aβ) peptide is considered a main mechanism responsible for neuronal death in Alzheimer’s disease. However, the molecular basis of this toxicity, in particular the interaction of Aβ and its aggregates with a cell membrane, remains unclear. Solidstate NMR (ssNMR) is a very well-suited technique for studies of the molecular basis of Aβ peptide interactions with cell membranes, but low sensitivity limits such studies due to a large fraction of the sample volume being taken up by lipids. Dynamic Nuclear Polarisation (DNP) allows increasing the signals in ssNMR experiments, thus enabling measurements with lower amounts of protein material. In this work, the feasibility of structural ssNMR-DNP studies of Aβ(1-40) peptide, at low concentration, interacting with biomimetic lipid bilayers was explored.
DNP enhancements of between 20-87 were observed for lipid and protein 13C spins under a range of conditions. Sample conditions were optimised to give maximum signal strength under magic angle spinning (MAS) DNP conditions. The interaction between biradical and glycerol cryoprotectant, required for efficient DNP, and lipid vesicles was probed. Biradical was shown to have a significant interaction with lipid vesicles, with glycerol causing a partitioning effect into lipid vesicles, determined by paramagnetic relaxation enhancement (PRE) experiments. 31P chemical shift anisotropy (CSA) patterns of lipid vesicles showed only slight changes on addition of biradical and glycerol, indicating no major changes in vesicle structure. Two-dimensional 13C13C (DARR) and 15N13C (NCA, NCACX) correlation experiments were possible in under 3 hours at lipid-to-peptide ratio (L:P) of 20:1. In order to reduce the contribution of signals from natural abundance 13C of lipids we used double quantum (DQ) experiments. POST-C7 double-quantum single-quantum (DQSQ) correlation was shown to be feasible at L:P of 100:1 (70 nmol peptide) and 200:1 (35 nmol peptide) with well resolved cross peaks in under 10 hours and under 20 hours, respectively. Structural Aβ(1-40) information was also obtained in a complex mixture of membrane mimicking lipids, including the ganglioside GM1 and cholesterol, which have been shown to modulate both Aβ and membrane properties. DNP enhancement of 87 was achieved for rat synaptic membrane extracts, and structural Aβ(1-40) information was obtained following addition and incubation with the membrane extracts. Secondary chemical shifts at the uniformly labelled amino acids in the Aβ(1-40) sequence agree with β-sheet conformation at these positions. This demonstrates that DNP enhanced ssNMR can be used to probe structures of Aβ(1-40) which exist at low concentrations in a cell membrane environment, as well as in physiologically relevant samples where membranes have been extracted from mammalian cells. This will lead to a better understanding of mechanism of cell membrane disruption by Aβ. Additionally, DNP-enhanced ssNMR measurements were made of Aβ(1-40) at two timepoints following addition to synthetic lipids, showing the feasibility of tracking kinetic Aβ events by flash freezing and recording spectra at cryogenic temperatures at various points in the process.
Dissolution Dynamic Nuclear Polarisation (d-DNP) is another method for generating strong NMR signals, but in solutions rather than solids. This is achieved by polarizing nuclear spins using microwave irradiation, followed by a rapid dissolution with hot solvent. The d-DNP setup at the University of Nottingham is based on a dual iso-centre magnet with its top section (3.4 T) used for polarizing the sample at low temperatures (~1.8 K), and the bottom section (9.4 T) used for solution NMR measurements at room temperature. The system was optimized to achieve 300 ms dead time (time between start of dissolution process and recording a signal) during which the sample is dissolved and transferred into an NMR tube. Many protein folding, unfolding and aggregation events develop on a similar timescale. The refolding process of hen egg-white lysozyme was used as a model, to which 19F-containing labels were introduced.
Maximum nuclear polarization was achieved with 40 mM water soluble TEMPOL as a polarizing agent in the glassy partially deuterated water/DMSO at the optimal microwave frequency of 94.05 GHz. Hen egg-white lysozyme was chemically modified to covalently attach trifluoroacetyl groups (CF3CO-) to six surface exposed amines of lysine residues. The protein was dissolved in a matrix consisting of 60/30/10 v:v DMSO-d6/D2O/H2O with 40 mM TEMPOL polarizing agent and 100 mM 5-fluorouracil, presence of which is shown to improve the enhancements of lysozyme. 900 ms after the dissolution, the NMR signals were acquired using a 90o pulse. The signal enhancements for 19F-nuclei of lysozyme and 5-fluorouracil were ~300 and ~1000 respectively, which show overall suitability of the approach. Polarisation of 19F- labelled lysozyme under protein denaturing conditions followed by dissolution into protein folding conditions and acquisition of DNP enhanced protein signals shows the feasibility of tracking kinetic protein events using this methodology.
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