| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Biochemistry and Molecular Biology, 2 Department of Public Health, and 3 National Institute for the Physics of Matter, University of Parma, 43100 Parma, Italy4 Department of Physics and National Institute for the Physics of Matter, University of Milan-Bicocca, 20126 Milan, Italy
(RECEIVED October 20, 2004; FINAL REVISION December 27, 2004; ACCEPTED January 3, 2005)
Many of the effects exerted on protein structure, stability, and dynamics by molecular crowding and confinement in the cellular environment can be mimicked by encapsulation in polymeric matrices. We have compared the stability and unfolding kinetics of a highly fluorescent mutant of Green Fluorescent Protein, GFPmut2, in solution and in wet, nanoporous silica gels. In the absence of denaturant, encapsulation does not induce any observable change in the circular dichroism and fluorescence emission spectra of GFPmut2. In solution, the unfolding induced by guanidinium chloride is well described by a thermodynamic and kinetic two-state process. In the gel, biphasic unfolding kinetics reveal that at least two alternative conformations of the native protein are significantly populated. The relative rates for the unfolding of each conformer differ by almost two orders of magnitude. The slower rate, once extrapolated to native solvent conditions, superimposes to that of the single unfolding phase observed in solution. Differences in the dependence on denaturant concentration are consistent with restrictions opposed by the gel to possibly expanded transition states and to the conformational entropy of the denatured ensemble. The observed behavior highlights the significance of investigating protein function and stability in different environments to uncover structural and dynamic properties that can escape detection in dilute solution, but might be relevant for proteins in vivo.
Keywords: molecular crowding; protein folding; protein immobilization; encapsulation; fluorescence
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041190805.
Reprint requests to: Stefano Bettati, Department of Public Health, University of Parma, Via Volturno 39, 43100 Parma, Italy; e-mail: stefano.bettati{at}unipr.it; fax: +29-0521-903712.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Vaitheeswaran and D. Thirumalai Interactions between amino acid side chains in cylindrical hydrophobic nanopores with applications to peptide stability PNAS, November 18, 2008; 105(46): 17636 - 17641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. Knotts IV, N. Rathore, and J. J. d. Pablo An Entropic Perspective of Protein Stability on Surfaces Biophys. J., June 1, 2008; 94(11): 4473 - 4483. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. D'Alfonso, M. Collini, F. Cannone, G. Chirico, B. Campanini, G. Cottone, and L. Cordone GFP-mut2 Proteins in Trehalose-Water Matrixes: Spatially Heterogeneous Protein-Water-Sugar Structures Biophys. J., July 1, 2007; 93(1): 284 - 293. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Baldini, F. Cannone, G. Chirico, M. Collini, B. Campanini, S. Bettati, and A. Mozzarelli Evidence of Discrete Substates and Unfolding Pathways in Green Fluorescent Protein Biophys. J., March 1, 2007; 92(5): 1724 - 1731. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Rathore, T. A. Knotts IV, and J. J. de Pablo Confinement Effects on the Thermodynamics of Protein Folding: Monte Carlo Simulations Biophys. J., March 1, 2006; 90(5): 1767 - 1773. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Cannone, S. Bologna, B. Campanini, A. Diaspro, S. Bettati, A. Mozzarelli, and G. Chirico Tracking Unfolding and Refolding of Single GFPmut2 Molecules Biophys. J., September 1, 2005; 89(3): 2033 - 2045. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Baldini, F. Cannone, and G. Chirico Pre-Unfolding Resonant Oscillations of Single Green Fluorescent Protein Molecules Science, August 12, 2005; 309(5737): 1096 - 1100. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
