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 protein, have shown that removal of five amino acids from the loop causes a dramatic reorganization of the wild-type structure. The new tetrameric molecule exhibits a significantly higher stability (Lassalle, M.W. et al., J. Mol. Biol., 1998, 279, 987-1000) and unfolds in a second order reaction (Lassalle, M.W. and Hinz, H.-J., Biochemistry, 1998, 37, 8465-8472). In the present investigation we report extensive refolding studies of RM6 at different temperatures and GdnHCl concentrations monitored by CD and fluorescence to probe for changes in secondary and tertiary structure, respectively. 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 deltaH#, deltaS# and deltaG# is very similar for unfolding and refolding. For both processes the activation properties approach a maximum in the vicinity of the denaturant concentration, c(K=1), where the equilibrium constant equals 1, i.e. deltaG0 equals zero. CD and fluorescence refolding kinetics are described by identical constants suggesting that the formation of secondary and tertiary structure occurs simultaneously. Refolding is, however, characterized by a more complex mechanism than unfolding. Although the general pattern is dominated by the sequence monomers to dimers to tetramers, 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.
Lassalle
M W
MW
Institut für Physikalische Chemie der Westfälischen Wilhelms-Universität, Münster, Germany.
Hinz
H J
HJ
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D019791:Guanidine; D007700:Kinetics; D017510:Protein Folding; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; C054243:Rop protein, ColE1 plasmid; D019791:Guanidine