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Relative and Regional Stabilities of the Hamster, Mouse, Rabbit, and Bovine Prion Proteins toward Urea Unfolding Assessed by Nuclear Magnetic Resonance and Circular Dichroism Spectroscopies

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Olivier Julien†, Subhrangsu Chatterjee†, Trent C. Bjorndahl‡, Braden Sweeting§

, Sandipta Acharya‡, Valentyna Semenchenko

, Avijit Chakrabartty§

#, Emil F. Pai§

, David S. Wishart‡

, Brian D. Sykes*†, and Neil R. Cashman@

Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7

Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada T6G 2E8

Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 2M9

The Campbell Family Cancer Research Institute, Ontario Cancer Institute, MaRS/TMDT, 101 College Street, Toronto, ON, Canada M5G 1L7

National Institute of Nanotechnology (NINT), National Research Council, 11421 Saskatchewan Drive, Edmonton, AB, Canada T6G 2M

Departments of Biochemistry and Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8

Department of Medicine (Neurology) and Brain Research Centre, University of British Columbia, Vancouver, BC, Canada V6T2B5

Biochemistry, 2011, 50 (35), pp 7536–7545

DOI: 10.1021/bi200731e

Publication Date (Web): July 29, 2011

Address: 4-19 Medical Sciences Building, Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7. Phone: (780) 492-5460. Fax: (780) 492-0886. E-mail: brian.sykes@ualberta.ca.

Funding Statement

This work was supported by the Alberta Prion Research Institute (funded by Alberta Ingenuity), PrioNet Canada (funded by the Networks of Centres of Excellence Canada Program and the Canadian Institutes of Health Research), and the Ontario Ministry of Health and Long-term Care. O.J. is the recipient of an AHFMR studentship and a Frederick Banting and Charles Best Canada Graduate Doctoral Scholarship from CIHR.

Author Contributions

O.J. and S.C. contributed equally to this work.

Section:

General Biochemistry

Abstract

The residue-specific urea-induced unfolding patterns of recombinant prion proteins from different species (bovine, rabbit, mouse, and Syrian hamster) were monitored using high-resolution ¹H nuclear magnetic resonance (NMR) spectroscopy. Protein constructs of different lengths, and with and without a His tag attached at the N-terminus, were studied. The various species showed different overall sensitivities toward urea denaturation with stabilities in the following order: hamster ≤ mouse < rabbit < bovine protein. This order is in agreement with recent circular dichroism (CD) spectroscopic measurements for several species [Khan, M. Q. (2010) Proc. Natl. Acad. Sci. U.S.A.107, 19808–19813] and for the bovine protein presented herein. The [urea]_1/2 values determined by CD spectroscopy parallel those of the most stable residues observed by NMR spectroscopy. Neither the longer constructs containing an additional hydrophobic region nor the His tag influenced the stability of the structured domain of the constructs studied. The effect of the S174N mutation in rabbit PrP^C was also investigated. The rank order of the regional stabilities within each protein remained the same for all species. In particular, the residues in the β-sheet region in all four species were more sensitive to urea-induced unfolding than residues in the α2 and α3 helical regions. These observations indicate that the regional specific unfolding pattern is the same for the four mammalian prion proteins studied but militate against the idea that PrP^Sc formation is linked with the global stability of PrP^C.

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