NR AOWP
AU McGuirl,M.A.; Chelmo,S.J.; Small,S.N.
TI Characterization of Tryptophan Mutants of Truncated (90-231) Hamster Prion Protein
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-69
PT Konferenz-Poster
AB
Prion diseases are characterized by an accumulation in the brain of a misfolded state of the prion protein, PrP. The C-terminal portion of PrP (residues 90-231) is essential to infectivity, has primarily A-helical structure and binds copper(II). The aggregated state of infectious PrP has stymied structural analyses, although there is a noted increase in B-sheet content upon conversion. However, soluble B-PrP forms may be produced in vitro from A-PrP.
The long-term goal of our research is to better define the structure of the soluble B-PrP forms. Our approach is to identify positions that undergo changes in local solvent environment upon conversion. We will identify these by monitoring the fluorescence emission spectra in a series of mutant PrP (90-231) proteins. We plan to substitute a single Trp codon at different locations in each variant of the prion gene. The mutant proteins will then be expressed under conditions amenable to the substitution of 7-AzaTrp for normal Trp. The 7-AzaTrp fluorescence emission spectrum is both unique compared with normal Trp and exquisitely sensitive to its local environment. Hence, we will detect structural differences between A- and B-PrP by monitoring fluorescence spectral changes as a function of the position of the 7-AzaTrp moiety.
In order to enhance the specificity and signal changes of the introduced 7-AzaTrp residue, we have prepared a Trp-free variant of PrP 90-231 as a base protein for site-directed mutagenesis. Residues 90-231 of hamster PrP contain two Trp residues, W99 and W145. Although W99 is strictly conserved, Tyr is often found at position 145 in other mammals. We report here a comparison of the A-helicity and stability of five recombinantly produced PrPs, in the presence and absence of copper: (a) wild type PrP (23-231), (b) wild type PrP(90-231), (c) PrP (90-231)/W99F, (d) PrP (90-231)/W145Y, and (e) the double mutant PrP (90-231)/W99F/W145Y.
AD M.A. McGuirl, S.J. Chelmo, S.N. Small, University of Montana, USA
SP englisch
PO Deutschland