Kinetics and mechanism of amyloid formation by the prion protein H1 peptide as determined by time-dependent ESR.
BACKGROUND
Peptides derived from three of four putative alpha-helical regions of the prion protein (PrP) form amyloid in solution. These peptides serve as models for amyloidogenesis and for understanding the alpha helix -->beta strand conformational change that is responsible for the development of disease. Kinetic studies of amyloid formation usually rely on the detection of fibrils. No study has yet explored the rate of monomer peptide uptake or the presence of nonfibrillar intermediate species. We present a new electron spin resonance (ESR) method for probing the kinetics of amyloid formation. A spin label was covalently attached to a highly amyloidogenic peptide and kinetic trials were monitored by ESR.


RESULTS
Electron microscopy shows that the spin-labeled peptide forms amyloid, and ESR reveals the kinetic decay of free peptide monomer during amyloid formation. The combination of electron microscopy and ESR suggests that there are three kinetically relevant species: monomer peptide, amyloid and amorphous aggregate (peptide aggregates devoid of fibrils or other structures with long-range order). A rather surprising result is that amyloid formation requires the presence of this amorphous aggregate. This is particularly interesting because PrPSc, the form of PrP associated with scrapie, is often found as an aggregate and amyloid formation is not a necessary component of prion replication or pathogenesis.


CONCLUSIONS
Kinetic analysis of the time-dependent data suggests a model whereby the amorphous aggregate has a previously unsuspected dual role: it releases monomer into solution and also provides initiation sites for fibril growth. These findings suggest that the beta-sheet-rich PrPSc may be stabilized by aggregation.
Lundberg
K M
KM
Department of Chemistry and Biochemistry University of California Santa Cruz, CA 95064, USA.
Stenland
C J
CJ
Cohen
F E
FE
Prusiner
S B
SB
Millhauser
G L
GL
D000682:Amyloid / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011328:Prions / Q000737:chemistry* / Q000648:ultrastructure; D013113:Spin Labels; D013997:Time Factors
D000682:Amyloid; D010455:Peptides; D011328:Prions; D013113:Spin Labels