Alzheimer's disease is associated with the abnormal deposit of amyloid beta protein (Ab) that occurs as fibrils within the cerebral neuropil. To characterize the way in which these fibrils assemble under different pH conditions, we have performed scanning transmission electron microscopy (STEM) on synthetic full-length Ab peptides as well as various Ab peptides with truncated sequences. STEM analysis of unstained preparations provides a quantitative determination of the mass-per-length (MPL) and thus the numbers of beta-sheets within fibrils. We have also investigated the use of inelastic dark-field imaging in the energy-filtering transmission electron microscope (EFTEM) as an alternative approach to determining the MPL of amyloid fibrils. Images were collected with an incident electron energy of 300 keV, and energy losses in the range 15 to 35 eV, which corresponds to the broad plasmon resonance for protein. The inelastic EFTEM mass maps were found to be of comparable quality to the STEM mass maps in terms of the signal to noise ratio, but the EFTEM images could be acquired more rapidly. In general, MPL measurements reveal the existence of fundamental fibrillizing units, or """"""""protofilaments,"""""""" consisting of well-defined numbers of cross-beta sheets. Variations in the arrangement of beta sheets in the amyloid fibrils at the EM level are being used by scientists in NIDDK to correlate with atomic-level information about the peptide backbone conformation obtained from NMR spectroscopy.
| Luca, Sorin; Yau, Wai-Ming; Leapman, Richard et al. (2007) Peptide conformation and supramolecular organization in amylin fibrils: constraints from solid-state NMR. Biochemistry 46:13505-22 |
