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Title: Characterising the folding intermediate and denatured state of the large β-barrel protein GFP
Author: Huang, J.-R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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A review of GFP discussing the folding studies to date has been published recently and is discussed in Chapter 1. In Chapter 3, the global and local stability of GFP was probed by measuring the H/D exchange rates of more than 157 assigned amide protons. Analysis of the exchange rates of amide protons of two different pHs allowed verification that amide protons with the β-barrel structure exchange at the EX3 limit, at which free energies of exchange can be calculated. Chemical denaturation was used to unfold the protein, changes in structure being monitored by optical probes (green fluorescence, tyrosine fluorescence, and circular dichroism) in Chapter 4. The results show that the denaturation behaviour of GFP is complex compared to many small proteins: equilibrium is established only very slowly, over the time courses of weeks, suggesting that there are high folding/unfolding energy barriers. H/D exchange rates over a wide range of conditions and as a function of denaturant concentration were undertaken. A super stable core is characterised in Chapter 5. Within this super stable core, there were seven amide hydrogens which resisted H/D exchange even at high pD (8.9), high temperature (37°C), and high concentrations of GdmCl (1.6 M). It is possible that the super stable core is the initiation site of the folding of GFP. Residual structure under denaturing conditions might play an important role in the folding pathway for GFP. The residual structure of the acid-denatured state of GFP has been characterised by far-UV CD, small angle X-ray scattering, and 19F-NMR techniques, whilst structural properties of this residual structure are still unknown. In Chapter 6 a variety of NMR techniques were applied to study the structure of the denatured state of GFP. These studies provide evidence of residual structure in the acid-denatured state.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available