Semisynthesis and application of homogeneous prion proteins with complex posttranslational modifications
Final Report Abstract
We have developed improved strategies for the semisynthesis of membrane-anchored Prion protein (PrP) variants based on soluble expression of intein fusion proteins in bacteria. The generated membrane-anchored PrP variants were extensively studied to gain insides into their conformational changes when attached to membranes and in solution. Using a variety of techniques such as fluorescence, CD and NMR spectroscopy we have been able to distinguish different binding modes of full-length PrP as well as of two mutants to negatively charged membranes that heavily depend on the presence of a membrane anchor mimicking the native glycosylphosphatidyl inositol (GPI) anchor. These different conformations help to explain conversion of cellular PrP into the predominantly β-sheet-rich PrP forms that are involved in pathogenicity of PrP. The occurrence of spontaneously forming membrane pores as observed with a specific PrP mutant hints towards cytotoxic effects of PrP by harming membrane integrity. Furthermore, new routes to glycosylated PrP isoforms have been developed relying on recent progress in peptide and protein chemistry. These glycosylated PrP variants will allow deciphering the impact on N-glycosylation on conformational stability and conversion of PrP.
Publications
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(2012) The effect of C-terminal membrane anchor on prion protein interactions with membranes. Prion Conference, Amsterdam, The Netherlands. Prion, 6, supplement: 117
Chu K.N., Araman M.C., Becker C.F.
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(2013) Recombinant expression of soluble murine Prion protein for C-terminal modification. FEBS Letters, 587, 430-435
Chu, N.K., Becker, C.F.
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(2014) A C-terminal membrane anchor affects the interactions of prion proteins with lipid membranes” J. Biol. Chem., 289, 30144–30160
Chu, N.K., Shabbir, W., Bove-Fenderson, E., Araman, C., Lemmens-Gruber, R., Harris, D.A., Becker, C.F.
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(2014) A semisynthetic approach to investigate the influence of posttranslational modifications on prion pathogenicity. Prion Conference 2014, Trieste, Italy
Araman M.C., Becker C.F.
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(2014) Studying Weak and Dynamic Interactions of Posttranslationally Modified Proteins using Expressed Protein Ligation, ACS Chem. Biol., 9, 347-352
Tripsianes, K., Chu, N.K., Friberg, A., Sattler, M., Becker, C.F.
