Protein Folding Studied by Site Directed Mutagenesis

Abstract: The N-terminal RNA binding domain of U1A has been shown to fold in a two-state process without accumulating any intermediates. U1A has a symmetrically curved chevron plot which seems to result mainly from changes of the transition state. At low concentrations of denaturant the transition state occurs early in the folding reaction, whereas at high denaturant concentration it moves close to the native structure. In paper I of this thesis, this movement is used to follow continously the formation and growth of U1A's folding nucleus by phi-value analysis. Although U1A's transition state structure is generally delocalised and displays a typical nucleation-condensation pattern, it is still possible to resolve a sequence of folding events. However, these events are sufficiently coupled to start almost simultaneously throughout the transition state structure. In paper II of this thesis, folding rates for the F30H mutant of protein G were measured by 15N nuclear spin relaxation under near native equilibrium conditions. Data of satisfactory precision were obtained for a subset of residues that together probe every secondary structure element in the protein. In general, the residue specific rates measured by NMR agree well with the rate obtained by stopped-flow fluorescence measurements. A single outlier with significantly slower folding rate was found in the N-terminal segment that is known to be unstructured in the acid denatured state, all other secondary structure elements partially populate native-like conformations in this state. The result may reveal a hierarchy of folding, and suggests that the folding rate dependes on the degree of native-like structure in the denatured state. Unfolding and inactivation of cutinase by AOT and guanidine hydrochloride (GdnHCl) was studied by equilibrium and time-resoved methods. The results shows that cutinase from F. solani and H. insolens unfolds in a cooperative two-state process in AOT and GdnHCl. However, linear extrapolation of the protein stability using equilibrium data gave significantly different values, which suggests that the denatured states in AOT and GdnHCl are different. Far-UV CD data reveals that the AOT denatured state retains some structure but the GdnHCl denatured state is essensially unstructured. The denatured state in AOT reversed micelles does not differ significantly from that observed in aqueous AOT. NMR characterisation of the denatured state in reversed micelles indicates a molten globule like state, consistent with the CD data.