| Summary: | Ubiquitin is a small globular protein (76 amino acids, MW 8565 Da) which occurs in both eukaryotes and prokaryotes. It is involved in many biological processes, its function to 'tag' other proteins, to signal degradation. Ubiquitin is an ideal model for structural studies since it has a well defined secondary and tertiary structure and is stable to changes in temperature, pH and to a wide range of solvents. Synthetic techniques have been used to incorporate unnatural amino acids into the hydrophobic core of ubiquitin, the aim being to observe the structural defects incurred by these changes. Two analogues of ubiquitin have been under investigation. The first analogue has the unnatural amino acids, 2S, 4S, 5-Fluoroleucine substituted into two leucine positions which, from studies of the native protein were directed towards each other, across the hydrophobic core. The structure of this fluorinated analogue has been determined using both <SUP>1</SUP>H and <SUP>19</SUP>F, 1D and 2D NMR methods. The NMR solution structure reveals that the fluoro-methyl groups adopt preferred conformations within the protein core. The second analogue incorporates unnatural aminobutyric acid and norvaline into positions on the α-helix, at positions i, i +4. These unnatural amino acids replace valine and isoleucine, therefore eliminating β-branching at these specific positions and also giving a net loss of two methyl volumes from the hydrophobic core. The structure has been determined using <SUP>1</SUP>H 2D NMR and detailed comparisons made to the X-ray crystallographic structure of the analogue. The solution structure reveals that the substituted residues adjust their positions to compensate for the lost methyl volume.
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