Refolding Studies on the Tetrameric Loop Deletion Mutant RM6 of ROP Protein
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M.W. Lassalle
Abstract
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 ∆H#, ∆S# and ∆G# 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. ∆G0 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.
Copyright © 1999 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Protein Kinase C-α Produces Reciprocal Effects on the Phorbol Ester Stimulated Tyrosine Phosphorylation of a 50 kDa Kinase in Jurkat Cells
- Growth- and Development-Dependent Expression of Gangliosides in Rat Hepatocytes and Liver Tissues
- Regulated Phosphorylation of the Gal4p Inhibitor Gal80p of <I>Kluyveromyces lactis</I> Revealed by Mutational Analysis
- The WW Domain of Dystrophin Requires EF-Hands Region to Interact with β-Dystroglycan
- Purification and MALDI-MS Characterization of Stressin, a Stress-Associated Glycoprotein
- Direct Selection of EGF Mutants Displayed on Filamentous Phage Using Cells Overexpressing EGF Receptor
- Refolding Studies on the Tetrameric Loop Deletion Mutant RM6 of ROP Protein
- Secretion of Protease Nexin-II/Amyloid β Protein Precursor by Human Colorectal Carcinoma Cells and Its Modulation by Cytokines/Growth Factors and Proteinase Inhibitors
- Isolation and Primary Structure of the CCI Papain-Like Cysteine Proteinases from the Latex of Carica candamarcensis Hook
- Identification of SLPI (Secretory Leukocyte Protease Inhibitor) in Human Mast Cells Using Immunohistochemistry and In Situ Hybridisation
- Stereochemistry of the Interaction between Methionine Sulfur and the Protein Core
- Trypsin from Pacifastacus leninsculus Hepatopancreas: Purification and cDNA Cloning of the Synthesized Zymogen
