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Title: Structure formation in N-terminal fragments of ubiquitin
Author: Bolton, D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2001
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The initial aim of this project was to shed light on early events in the folding of ubiquitin by studying two N-terminal fragments thought to be partially structured in the proposed ubiquitin folding intermediate. Several peptide constructs were made by a variety of molecular biology approaches. The most efficient and versatile method proved to be expression as a GST fusion protein followed by cleavage with thrombin and purification by reverse phase HPLC. The peptide fragments UQ(I-5I) and UQRH formed novel, structured aggregates under physiological conditions (50 mM sodium phosphate, pH 7) and as such cannot be used as models of the physiologically unfolded state of ubiquitin. However, both peptides displayed interesting properties, which became the subject of subsequent investigation. UQ(I-5I) encompasses strands, U1, U2 the α-helix, and strand U3 of native ubiquitin. In low concentrations of sodium sulphate UQ(I-5I) behaves like a "molten-globule" adopting many different conformations, whilst in high concentrations of sodium sulphate the peptide has a unique dimeric structure. The solution structure of UQ(I-5I) in 0.8 M sodium sulphate was determined by triple resonance NMR methods. This indicates how the peptide can dimerise and fold, with some elements of the new structure being similar to ubiquitin (such as the helix-helix interface) and others non-native (for example the packing of strand U3 at the dimer interface). Analysis of UQ(I-5I) under lower concentrations of sodium sulphate gave insight into the causes of its structural heterogeneity. The properties of the minor forms populated under these conditions, suggest that the structural heterogeneity arises from a segmental motion of strand U3 in a single subunit of the UQ(I-5I) dimer.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available