Refolding studies on the tetrameric loop deletion mutant RM6 of ROP protein

Previous DSC and X-ray studies on RM6, a loop deletion mutant of wtROP prot ein, have shown that removal of five amino acids from the loop causes a dra matic reorganization of the wild-type structure, The new tetrameric molecul e exhibits a significantly higher stability (Lassalle, M.W. et al., J. Mel. Biol,, 1998, 279, 987-1000) and unfolds in a second order reaction (Lassal le, M.W. and Hint, H.-J., Biochemistry, 1998, 37, 8465-8472). In the present investigation we report extensive refolding studies of RM6 a t different temperatures and GdnHCl concentrations monitored by CD and fluo rescence to probe for changes in secondary and tertiary structure, respecti vely. The measurements permitted us to determine activation parameters as a function of denaturant concentration. The results demonstrate convincingly that the variation with GdnHCl concentration of the activation parameters Delta H-#, Delta S-# and Delta G(#) is very similar for unfolding and refol ding. For both processes the activation properties approach a maximum in th e vicinity of the denaturant concentration, C-(K=1), where the equilibrium constant equals 1, i.e. Delta G(0) equals zero. CD and fluorescence refoldi ng kinetics are described by identical constants suggesting that the format ion of secondary and tertiary structure occurs simultaneously. Refolding is , however, characterized by a more complex mechanism than unfolding. Althou gh the general pattern is dominated by the sequence monomers to dimers to t etramers, parallel side reactions involving dimers and monomers have to be envisaged in the initial folding phase, supporting the view that the native state of RM6 can be reached by several rather than a single pathway.