A Synchrotron-based Hydroxyl Radical Footprinting Analysis of Amyloid Fibrils and Pre-fibrillar Intermediates with Residue-specific Resolution.

Nov 9, 2014

Structural models of the fibrils formed by the 40-residue amyloid-beta (Ab40) peptide in Alzheimer's Disease typically consist of linear polypeptide segments, oriented approximately perpendicular to the long axis of the fibril, and joined together as parallel in-register b-sheets to form filaments. However, various models differ in the number of filaments that run the length of a fibril, and in the topological arrangement of these filaments. In addition to questions about the structure of Ab40 monomers in fibrils, there are important unanswered questions about their structure in pre-fibrillar intermediates, which are of interest because they may represent the most neurotoxic form of Ab40. To assess different models of fibril structure, and to gain insight into the structure of pre-fibrillar intermediates, the relative solvent accessibility of amino acid residue side chains in fibrillar and pre-fibrillar Ab40 preparations were characterized in solution by hydroxyl radical footprinting and structural mass spectrometry. A key to the application of this technology was the development of hydroxyl radical reactivity measures for individual side-chains of Ab40. Combined with mass-per-length measurements performed by darkfield electron microscopy, the results of this study are consistent with the core filament structure represented by two- and three-filament solid state NMR based models of the Ab40 fibril (such as 2LMN/O/P/Q), with minor refinements, but they are inconsistent with the more recently proposed 2M4J model. The results also demonstrate that individual Ab40 fibrils exhibit structural heterogeneity or polymorphism, where regions of two-filament structure alternate with regions of three-filament structure. The footprinting approach utilized in this study will be valuable for characterizing variant forms of the Ab peptide in vitro and in vivo.

Figure: Cross-sections of two different three-filament fibril models with van-der-Waals spheres for non-hydrogen atoms. Below each model is a ball-and-stick rendering illustrating the backbone conformation of one filament in the model. LEFT: The 2LMP model with the three Y10 residues at the N-termini labeled. Note that M35 (marked by * in the lower filament) is exposed to the water-filled central channel, consistent with the low observed protection factor. Also note that residues F20 and I31 appear to be much more solvent accessible than their adjacent residues of the same type (F19 and I32), consistent with the observed protection factors. All other residues for which data is available are either buried or accessible in this model, consistent with the measured protection factors. RIGHT: the 2M4J model with its three N-terminal "D1" residues marked. Note that the backbone conformation does not correspond to a b-sheet, and there are numerous discrepancies between expected and observed protection factors. For example, F19 and F20 appear to be similarly buried, contrary to the observed protection factors. Also, I31 is buried while I32 is not, the converse of the relationship indicated by the observed protection factors. M35 (marked by * in the lower filament) appears to be lessaccessible than V40 in this model, again the converse of the relationship indicated by the observed protection factors.

Results from: Klinger AL, Kiselar J, Ilchenko SA, Komatsu H, Chance MR, Axelsen PH. Biochemistry. 2014 Nov 9. [Epub ahead of print]