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M400952200v1
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Papers In Press, published online ahead of print March 18, 2004
J. Biol. Chem, 10.1074/jbc.M400952200
Submitted on January 28, 2004
Revised on March 16, 2004
Accepted on March 18, 2004

Characterization of recombinant, membrane-attached full-length prion protein

Heike Eberl, Peter Tittmann, and Rudi Glockshuber

Biology Dept., Institute of Molecular Biology and Biophysics, Zurich CH-8093

Corresponding Author: rudi{at}mol.biol.ethz.ch

An abnormal isoform, PrPSc, of the normal, cellular prion protein, PrPC, is the major component of the causative agent of prion diseases. Both isoforms were found to possess the same covalent structures, including a C-terminal GPI-anchor, but different secondary and tertiary structures. In this study, a variant of full-length PrP with an unpaired cysteine at the C-terminus was recombinantly produced in Escherichia coli, covalently coupled to a thiol-reactive phospholipid, and incorporated into liposomes to serve as model for studying possible changes in structure and stability of recombinant PrP upon membrane attachment. Covalent coupling of PrP to liposomes did not result in significant structural changes observable by far-UV circular dichroism. Moreover, limited proteolysis experiments failed to detect changes in stability of liposome-bound PrP relative to soluble PrP. These data suggest that the requirement of raft localization for the PrPC to PrPSc conversion, previously observed in cell culture models, is not due to a direct influence of raft lipids on the structure and stability of membrane bound PrPC, but caused by other factors, e.g. increased local PrP concentrations or high effective concentrations of membrane-associated conversion factors. The availability of recombinant PrP covalently attached to liposomes provides the basis for systematic in vitro conversion assays with PrP on the surface of membranes. In addition, our results indicate that the three-dimensional structure of mammalian PrPC in membranes is identical to that of recombinant PrP in solution.


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