Synthesis of double-fluorescent labeled prion protein for FRET analysis

Abstract

An abnormal form of prion protein (PrP) is considered to be the pathogen in prion diseases. However, the structural details of this abnormal form are not known. To characterize the non-native structure of PrP, we synthesized position-specific double-fluorescent labeled PrP for a fluorescence resonance energy transfer (FRET) experiment. Using FRET, we observed a conformational change in the labeled PrP associated with amyloid fibril formation. The FRET analysis indicated that the distance between fluorescent labeled N- and C-terminal sites of PrP increased upon the formation of amyloid fibrils compared with that of the native state. This approach using FRET analysis is useful for elucidating the structure of abnormal PrP.

Graphical abstract

The double-fluorescent labeled PrP is a useful for analyses of the structure and the formation of amyloid fibrils or PrP^Sc by using FRET.

Key words:

• prion protein
• four-base codon
• cell-free translation
• fluorescence resonance energy transfer (FRET)
• amyloid fibril

Acknowledgments

The coordinates of the model PrP^Sc structure were kindly provided by Cédric Govaerts. We thank Ryoji Abe and Kazuhide Imai of ProteinExpress Co., Ltd., for their assistance with the preparation of and experiments with double-labeled proteins.

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes

Abbreviations: CD, circular dichroism; EM, electron microscope; FRET, fluorescence resonance energy transfer; HXMS, hydrogen/deuterium exchange mass spectrometry; PrP, prion protein; PrP^Sc, scrapie form of prion protein; PrP^C, cellular form of prion protein; B558AF, BODIPY558-linked p-amino-L-phenylalanine; BFLAF, BODIPYFL-linked p-amino-L-phenylalanine; mPrP(121–231), mouse prion protein of amino acid residues 121–231; mPrP(23–231), mouse prion protein of amino acid residues 23–231.

Additional information

Funding

This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan, Grant-in-Aid for Scientific Research [grant number 21790441].