| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA
2 Center for Fluorescent Spectroscopy, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA
3 Institute for Neurodegenerative Diseases, Department of Neurology and
4 Institute for Neurodegenerative Diseases, Departments of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143, USA
(RECEIVED September 24, 2003; FINAL REVISION October 31, 2003; ACCEPTED November 10, 2003)
-helical isoform to a 
-sheet-rich isoform underlies the pathogenesis of sporadic prion diseases. To study the rate-limiting steps of spontaneous conversion, the formation of amyloid fibrils by the recombinant human PrP C-terminal fragment spanning residues 90–231 (recPrP) was monitored in the presence of urea. The kinetics of spontaneous fibril formation displayed sigmoidal behavior involving a lag phase. The shortest lag phase was observed at partially denaturing conditions, close to the concentration of urea corresponding to the middle point of unfolding. This result indicates that unfolding intermediates may be important for the conversion. To test whether unfolding intermediates are formed, we employed size-exclusion chromatography and circular dichroism spectroscopy to monitor urea denaturation of recPrP. Both techniques showed a single sigmoidal transition with very similar thermodynamic parameters of denaturation and that the transition can be described by a simple equilibrium between folded and denatured states. Detailed analyses of data, however, revealed that the dimensions of both the native and denatured species gradually increases with urea. Expansion of the native species is also accompanied by an increase in efficiency of the energy transfer from a single Trp residue to 1-anilinonaphthalene-8-sulfonate dye as measured by fluorescence. These data illustrate that thermodynamic character of the native ensemble changes gradually with environmental conditions. Such behavior is consistent with the thermodynamically variable model, and may be linked to the ability of PrP to adopt distinct abnormal conformations under pathologic conditions. Keywords: prion protein; conformational transition; amyloid fibrils; unfolding intermediates; size exclusion chromatography
Abbreviations: 1 PrPC, cellular isoform of the prion protein • PrPSc, disease-associated isoform of the prion protein • recPrP, recombinant prion protein • -recPrP, -helical monomeric form of recPrP • ANS, 1-anilinonaphthalene-8-sulfonate • CD, circular dichroism • recPrP106, recombinant prion protein of 106 residues • ThT, Tioflavin T • GndHCl, guanidine hydrochloride • SEC, size-exclusion chromatography
Reprint requests to: Ilia V. Baskakov, Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 W. Lombard Street, Baltimore, MD 21201, USA; e-mail: Baskakov{at}umbi.umd.edu; fax: (410) 706-8184.
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03457204.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Makarava and I. V. Baskakov The Same Primary Structure of the Prion Protein Yields Two Distinct Self-propagating States J. Biol. Chem., June 6, 2008; 283(23): 15988 - 15996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Sun, L. Breydo, N. Makarava, Q. Yang, O. V. Bocharova, and I. V. Baskakov Site-specific Conformational Studies of Prion Protein (PrP) Amyloid Fibrils Revealed Two Cooperative Folding Domains within Amyloid Structure J. Biol. Chem., March 23, 2007; 282(12): 9090 - 9097. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Gerber, A. Tahiri-Alaoui, P. J. Hore, and W. James Oligomerization of the Human Prion Protein Proceeds via a Molten Globule Intermediate J. Biol. Chem., March 2, 2007; 282(9): 6300 - 6307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Tahiri-Alaoui, V. L. Sim, B. Caughey, and W. James Molecular Heterosis of Prion Protein beta-Oligomers: A POTENTIAL MECHANISM OF HUMAN RESISTANCE TO DISEASE J. Biol. Chem., November 10, 2006; 281(45): 34171 - 34178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. N. Frankenfield, E. T. Powers, and J. W. Kelly Influence of the N-terminal domain on the aggregation properties of the prion protein Protein Sci., August 1, 2005; 14(8): 2154 - 2166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Novakofski, M. S. Brewer, N. Mateus-Pinilla, J. Killefer, and R. H. McCusker Prion biology relevant to bovine spongiform encephalopathy J Anim Sci, June 1, 2005; 83(6): 1455 - 1476. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Tahiri-Alaoui and W. James Rapid formation of amyloid from {alpha}-monomeric recombinant human PrP in vitro Protein Sci., April 1, 2005; 14(4): 942 - 947. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Kim, A. Muresan, K. Y. C. Lee, and R. M. Murphy Urea modulation of {beta}-amyloid fibril growth: Experimental studies and kinetic models Protein Sci., November 1, 2004; 13(11): 2888 - 2898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bozzi, E. Di Stasio, D. O. Cicero, B. Giardina, M. Paci, and A. Brancaccio The effect of an ionic detergent on the natively unfolded {beta}-dystroglycan ectodomain and on its interaction with {alpha}-dystroglycan Protein Sci., September 1, 2004; 13(9): 2437 - 2445. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
