Molecular structures of diffusible amyloid intermediates, commonly observed in misfolding of amyloid proteins into fibrils, have attracted broad interest because these intermediates are potent neurotoxins that may be responsible for amyloid diseases such as Alzheimer's disease (AD) and because structures of the intermediates would provide new insight into the misfolding pathway. However, owing to the intrinsically unstable, heterogeneous, and non-crystalline nature of the intermediates, traditional approaches, such as X-ray crystallography and solution NMR, have been ineffective for elucidating their molecular structures. The long-term objective of this research is to determine the structural changes of amyloid proteins in the course of misfolding into fibrils by solid-state NMR (SSNMR). The underlying problems hindering structural determination of amyloid intermediates are twofold (1) lack of effective protocols to isolate a specific transient intermediate, and (2) lack of methods to determine site-resolved structures. ? ? We have developed a novel approach using SSNMR that will permit the site-resolved structural determination of intermediates in fibril formation for a 40-residue Alzheimer's beta-amyloid peptide, Abeta(1-40). In this approach, conformational and morphological changes of the amyloid peptide are detected by optical spectroscopy and electron microscopy (EM). Then, quantitative structural examination is performed for freeze-trapped intermediates by SSNMR, which has been a dominant method for structural analysis of thermally stable amyloid fibrils. We hypothesize that in misfolding of Abeta(1-40), a diffusible intermediate containing beta-sheets exists prior to fibrillization. Based on the hypothesis, we will detect the soluble beta-sheet intermediates at reduced temperature, which slows fibril formations, using EM and fluorescence in the presence of a thioflavin T (ThT) dye, an indicator of beta-sheet-rich aggregates. The misfolding kinetics will be further examined by analysis of incubation-time dependence of EM images, CD spectra, and ThT fluorescence. Then, we will determine site-resolved secondary structures and supramolecular structures for the beta-sheet intermediate of Abeta(1-40), which is freeze trapped and subsequently lyophilized, by various SSNMR methods. These measurements will test our hypothesis that a spherical amyloid intermediate of 15-30 nm in diameter exists prior to fibrillization of Abeta(1-40) and that the intermediate contains well-ordered beta-sheets in the C-terminal and hydrophobic core. We will also examine kinetics and site-resolved intermediate structures for an E22G mutant of Abeta(1-40), which is known to stabilize subfibrillar intermediates. For this mutant, we will isolate two insoluble spherical intermediates in diameters of approximately 20 nm and 20-30 nm, both of which contain beta-sheet structures. The locations of the beta-sheet regions in the E22G intermediates will be further identified by SSNMR. ? ? ?
