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Title: Biomolecular imaging at high spatial and temporal resolution in vitro and in vivo
Author: Sharp, Thomas Harry
ISNI:       0000 0004 2718 2561
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2012
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This Thesis covers two separate projects linked by transmission electron microscopy (TEM) of biological samples: firstly, the utilisation of cryoTEM- TEM performed at cryogenic temperatures-to elucidate the superstructure of self-assembled peptide fibres, and secondly the development of new probes for Correlative Light Electron Microscopy (CLEM) both in vitro and in vivo. CryoTEM involves imaging samples in a hydrated state as close to experienced in bulk solvent as possible. This yields images and data that reflect the native state, and different to those of negatively stained samples that have been dried down and stained. Self-assembling peptide fibres (SAFs) were developed in the Woolfson lab in 2000 and characterised as α-helical coiled coils that grow both longitudinally and laterally. To date, data suggests an ordered superstructure, but negative stain TEM images and fibre X-ray diffraction have yielded only low resolution (~20 A) information. In this Thesis, I show that SAFs subjected to rapid plunge freezing and cryoTEM display a remarkable superstructure. Individual micrographs give high- resolution data that allowed direct structural interpretation of the packing of individual α-helices within the fibre, and the construction of a 3D electron density map at 8 A resolution. Furthermore, an all-atom model was derived combining the cryoTEM data and a 2.3 A X-ray crystal structure of a variant of the building block incapable of forming fibres. Together these provide the highest-resolution structure of a de novo designed protein-based supramolecular fibre. Green fluorescent protein (GFP) has revolutionised molecular and cell biology by enabling visualisation of endogenous proteins within live cells, providing information on protein motility, localisation and interactions. How- ever, the resolution of the light microscope (LM) is inherently limited by the wavelength of visible light, a problem that can be overcome by EM. However, EM requires a fixed cell. Correlative light electron microscopy (CLEM) combines the advantages of both techniques to allow visualisation of proteins in the live cell prior to detailed analysis at high resolution in the EM. At present there is no genetically encoded monomeric protein that is visible in both LM and EM. Therefore, the aim of the second project was to design and characterise a protein that was both fluorescent and electron dense. To achieve this, concatenation of a metal-binding protein, metallothionein (MT), was used to cluster heavy-metal ions into electron-dense nanoparticles. This was fused to Enhanced-GFP (EGFP) and characterised as a general-purpose "clonable" tag for use in CLEM. Metal binding capabilities were probed with mass spectrometry and dynamic light scattering. In vitro localisation of the probe in the EM was achieved by encapsulation within liposomes and fusion to SNXl, a membrane tabulating protein. In vivo, the probe was fused to various intra- and extracellular localisation domains and whole cell CL EM attempted. The probe proved difficult to visualise, oven after autometallography with gold. So, a separate complementary tag was also designed. A mutated intracellular protein, FK506 Binding Protein (FKBP12(F36V)), was fused to EGFP. FKBP12(F36V) has high affinity for a novel ligand (SLF'), which can be linked to a single gold particle and delivered to the cytoplasm. This would yield a fluorescent protein with an electron-dense particle nonco- valently bound. Preliminary studies are described towards this second goal, and results so far appear promising.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available