Biophysical studies of the green fluorescent protein

• Main Author: Craggs, T. D.
• Published: University of Cambridge 2007
• Subjects: 571.4
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598115

Chemical denaturation was used to unfold the protein, changes in structure being monitored by the green fluorescence. The denaturation behaviour of GFP was found to be complex compared to many small proteins: equilibrium was established only very slowly, over the time course of weeks, suggesting the existence of high (un)folding energy barriers. Kinetic experiments confirmed that the rates of unfolding at low concentrations of denaturant were very low, consistent with the system being slow to reach equilibrium. A rigorous quantitative analysis was used to reveal the presence of a populated intermediate. A change in the rate determining step to unfolding was observed with increasing denaturant concentrations, consistent with the existence of an unfolding intermediate. A spectroscopic characterisation of the acid and GdmC1 denatured states indicated that some residual structure persists under certain denaturing conditions. ¹⁹F-NMR techniques were employed to gain further insight into the structure and dynamics of the native and denatured states of 3-fluorotyrosine labelled GFP. Protein engineering was used systematically to replace each of the tyrosines enabling the full assignment of the ¹⁹F-NMR spectrum. ¹⁹F photo-CIDNP (chemically induced dynamic nuclear polarisation) techniques were then used to characterise the native and denatured states of GFP. A strong correlation was established between the observed CIDNP effect and the solvent accessibility of the highest occupied molecular orbital of the tyrosines. This work has paved the way for real-time kinetic unfolding studies. Finally, novel techniques were developed which allow the equilibrium and kinetic characterisation of (un)folding pathways at the single-molecule level. This new methodology enabled the direct detection of the unfolding intermediate of Citrine, a yellow variant of GFP.