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J. Biol. Chem., Vol. 279, Issue 24, 25058-25065, June 11, 2004

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Characterization of Recombinant, Membrane-attached Full-length Prion Protein^*

Heike Eberl, Peter Tittmann, and Rudi Glockshuber¶

From the Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg and Elektronenmikroskopie-Zentrum der ETH Zürich, Institut für Angewandte Physik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland

An abnormal isoform, PrP^Sc, of the normal cellular prion protein (PrP^C) 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 glycosylphosphatidylinositol 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 a 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 the stability of liposome-bound PrP relative to soluble PrP. These data suggest that the requirement of raft localization for the PrP^C to PrP^Sc conversion, observed previously in cell culture models, is not because of a direct influence of raft lipids on the structure and stability of membranebound PrP^C 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 recombinant PrP on the surface of membranes. In addition, our results indicate that the three-dimensional structure of mammalian PrP^C in membranes is identical to that of recombinant PrP in solution.

Received for publication, January 28, 2004 , and in revised form, March 16, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland. Tel.: 41-1-6336819; Fax: 41-1-6331036; E-mail: rudi{at}mol.biol.ethz.ch.

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