Confomational analysis of soluble oligomers of GFP tagged prion protein by fluorescence fluctuation spectroscopy.
The conversion of prion protein (PrP) from the monomeric cellular isoform to the oligomeric pathological isoform is a crucial event in the pathogenesis of prion diseases. To investigate oligomer formation of PrP, enhanced green fluorescent protein (EGFP)-tagged PrP (EGFP-PrP) without the glycosylphosphatidylinositol (GPI) anchor was prepared and the oligomerization of EGFP-PrP induced by sodium dodecyl sulphate (SDS) was monitored by fluorescence correlation spectroscopy (FCS). The FCS analysis indicated that soluble oligomers were formed at 0.011% SDS. Furthermore, the combination of fluorescence cross-correlation spectroscopy (FCCS) and a panel of anti-PrP monoclonal antibodies (mAbs) revealed the conformational changes in PrP. Our studies provide a method to analyze conformational changes of proteins in solution.
Sakata
Hiroshi
H
Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
Horiuchi
Motohiro
M
Takahashi
Izumi
I
Kinjo
Masataka
M
D049452:Green Fluorescent Proteins / Q000737:chemistry*; D011328:Prions / Q000737:chemistry* / Q000648:ultrastructure*; D011487:Protein Conformation; D012995:Solubility; D013050:Spectrometry, Fluorescence / Q000379:methods*
D011328:Prions; D049452:Green Fluorescent Proteins