Characterization of Wild-type Aβ42 Globulomers We prepared Aβ42 oligomers with a globulomer protocol (13), which uses low concentration of SDS (0.05%) to mimic the lipid environment in the cell (42). TEM shows that the oligomer preparation contains mostly globular structures (Fig. 1A). Most globular oligomers have diameters ranging from 5 to 10 nm, with the majority of them around 7–8 nm (Fig. 1B). Elongated structures with a beaded string morphology can also be observed. The morphologies are similar to previous atomic force microscopy studies on globulomers, which show heights of 4–5 nm (35). MTT-based cell survival assays show that these Aβ42 oligomers are toxic to both PC-12 neuronal cells and HeLa cells in a dose-dependent manner (Fig. 1C). Approximately 20% of the cells died in the presence of 0.2 μm (monomer equivalent) Aβ42 globulomers, and ∼60% of cells died in the presence of 2 μm Aβ42 globulomers. The CD spectrum shows a major negative peak at 216–218 nm, indicative of mostly β-structures (Fig. 1D), and it is similar to Aβ42 fibrils (Fig. 1D). In comparison, the Aβ42 monomer shows a single large negative peak at around 198 nm, characteristic of random coil structures (Fig. 1D). X-ray powder diffraction reveals three major diffraction peaks at 4.2, 4.7, and 10 Å (Fig. 1E). The 4.7 Å reflection arises from the separation of strands within the same β-sheet. The peak at ∼10 Å corresponds to separations between β-sheets. The diffuse nature of the 10 Å peak suggests high heterogeneity of the intersheet packing. The origin of the 4.2 Å reflection is not well understood. Previously, an x-ray powder diffraction study of porcine stomach mucin protein showed a 4.2 Å reflection corresponding to distorted antiparallel β-structures (43), suggesting that similar structures may be present in Aβ42 globulomers. A SEC profile shows that the globulomers contain a major peak with a molecular mass of ∼100–150 kDa (Fig. 1F). A minor peak at >700 kDa likely corresponds to the species with beaded string morphology under TEM (Fig. 1A). SEC also shows that Aβ42 globulomers do not contain a significant amount of monomer or small oligomers (Fig. 1F). View larger version: Download as PowerPoint Slide FIGURE 1. Characterization of wild-type Aβ42 globulomers. A, TEM image of Aβ42 globulomers. B, diameters of Aβ42 globulomers from TEM studies. C, survival of PC-12 and HeLa cells in the presence of Aβ42 globulomers using MTT-based cell viability assay. The buffer control has the exact composition as in the final step of globulomer preparation. Error bars indicate S.D. D, CD measurement of Aβ42 globulomers, fibrils, and monomers. A major negative peak at 216–218 nm suggests that Aβ42 globulomers contain predominantly β-structures. E, x-ray powder diffraction of lyophilized Aβ42 globulomers. F, analytical size exclusion chromatography profile of Aβ42 globulomers. Solid trace, globulomers; dotted trace, protein standards. The molecular masses of protein standards are: peak 1, thyroglobulin, 670 kDa; peak 2, bovine γ-globulin, 158 kDa; peak 3, chicken ovalbumin, 44 kDa; peak 4, equine myoglobin, 17 kDa; peak 5, vitamin B12, 1.35 kDa.

Preparation of Spin-labeled Aβ42 Globulomers for EPR Study To study the structure of Aβ42 globulomers by EPR, we introduced spin labels, one at a time, at 14 residue positions throughout the Aβ42 sequence (Fig. 2A). The spin label side chain used in this study is named R1 (Fig. 2B). TEM studies on selected spin-labeled Aβ42 globulomer samples show mostly globular structures without fibrils or protofibrils (Fig. 2C), suggesting that spin labeling does not significantly perturb the formation of Aβ42 globulomers. View larger version: Download as PowerPoint Slide FIGURE 2. Globulomers of spin-labeled Aβ42. A, amino acid sequence of Aβ42 with spin labeling positions shown with arrowheads. B, structure of the spin label side chain R1 used in this work. C, TEM images of four representative spin-labeled Aβ42 globulomers show globular structures as major species.

Spin Label Mobility Analysis in Aβ42 Globulomers The EPR spectra of spin-labeled Aβ42 globulomers are shown in Fig. 3A (black traces). For spin label mobility analysis, we prepared the oligomers using a mixture of spin-labeled Aβ42 with wild-type Aβ42 at 1:3 molar ratio. Therefore, in the oligomer sample, only 25% of the Aβ42 molecules were labeled, and this will greatly reduce dipolar interactions between spin labels. Spin-spin interactions may broaden the EPR spectra and complicate the mobility analysis. View larger version: Download as PowerPoint Slide FIGURE 3. Residue-specific EPR mobility analysis for spin-labeled Aβ42 globulomers. A, the experimental EPR spectra of 25% labeled Aβ42 globulomers, which consist of spin-labeled and wild-type Aβ42 at 1:3 molar ratio, are shown in black. The best nonlinear least squares fits from spectral simulations are shown in red. All the fits contain two spectral components: a slow component (magenta) and a fast component (blue). B, plot of correlation time for the slow and fast components obtained from spectral simulations. C, plot of the populations of the fast and slow components from spectral simulations. D, residue-level local structural stability calculated using the relative populations of the slow (for structured state) and fast (for disordered state) components. Error bars in B and C indicate fitting errors. The site-specific spin label mobility is an indicator of residue-level structural order (44). The EPR spectra of spin-labeled Aβ42 globulomers encode information about the mobility of the spin label at each labeling site. The spin label mobility can be measured using center line width (45) or the ratio of high field and center line amplitude (46). For EPR spectra with multiple components reflecting multiple motional states of the spin label, however, these measurements report only the combined mobility from different spin label states. A quantitative method to extract mobility information is spectral simulation. Multiple spectral components can be simulated simultaneously, and the population of each component can be obtained from simulation (47). Therefore, we performed nonlinear least squares fitting to the EPR spectra using spectral simulation (see “Experimental Procedures”). The best fits are shown in Fig. 3A (red traces). Spectral fitting revealed two spectral components at every spin labeling position: a fast component and a slow component (Fig. 3A, magenta and blue traces). The presence of two spectral components suggests that globulomers contain at least two structural states whose interconversion is slower than the nanosecond timescale. The slow component has a correlation time of 2.0–3.0 ns in the N-terminal region and 3.0–4.5 ns in the C-terminal region, corresponding to a structured state. Spin labels at positions 1 and 4 have correlation time of ∼2.1 ns. Although they are the most flexible region in the Aβ42 sequence, the spin label mobility at these residues is comparable with ordered helix surface sites (44) or solvent-exposed β-sheet sites (48, 49). Therefore, the EPR data suggest that the N-terminal region adopts some ordered structures. The structural order increases from N terminus to C terminus. The most ordered region spans residues 32–40, with a correlation time between 3.5 and 4.5 ns. These residues have spin label mobility comparable with spin labels located in the hydrophobic core of globular proteins (50). The correlation time of the fast component is in the range of 0.7–1.7 ns, with decreasing side chain mobility from N- to C-terminal regions (Fig. 3B). Spin labels in completely unfolded proteins have a correlation time of ∼0.5 ns (46, 47), suggesting that this fast component corresponds to a partly disordered state. The relative population of the fast component is ∼25% for N-terminal region (residues 1–10) and 10–15% for most other residues (Fig. 3C), suggesting that the fast component represents a locally disordered state rather than a disordered monomer. This notion is further supported by the absence of a significant amount of monomer or small oligomer peaks in the SEC profile (Fig. 1F). The residue-level local stability can be calculated with the assumption that the structured state (corresponding to the slow component) and the locally disordered state (corresponding to the fast component) are in equilibrium. The free energy of local unfolding in Aβ42 globulomers is calculated using the percentages of slow and fast components from simulation (Fig. 3D). This local stability plot reveals three low stability regions: Asp-1–Tyr-10, Ser-26–Gly-29, and Gly-37. The low stability at residue Gly-37 supports a likely turn structure at this position. We investigated the temporal stability of Aβ42 globulomers using the spin label as a structural probe. The EPR spectral line shape of Aβ42 globulomers spin-labeled at residue 34 remains extremely similar upon incubation at room temperature (∼21 °C) for 0, 4, 8, 24, and 48 h (Fig. 4A), suggesting no structural changes during this incubation period. The results also show that there are no structural changes during EPR measurements, which typically take less than 20 min. View larger version: Download as PowerPoint Slide FIGURE 4. Temporal stability of Aβ42 globulomers. A, EPR spectra of Aβ42 L34R1 globulomers at room temperature in preparation buffer (5 mm phosphate, pH 7.4, 35 mm NaCl, 2% DMSO, and 0.05% SDS). B, EPR spectra of Aβ42 L34R1 globulomers at room temperature after 10-fold dilution to cell culture medium, which is ATCC-formulated RPMI 1640 medium with 10% heat-inactivated horse serum and 5% fetal bovine serum. Dotted line was drawn to aid comparison of different spectra. Next we studied the temporal stability of Aβ42 globulomers upon dilution into cell culture medium. Following a 10-fold dilution to RPMI 1640 medium, the EPR spectral line shape of Aβ42 globulomer remains largely the same, consisting of two spectral components (Fig. 4B). The line shape of both the ordered and the disordered components remains unchanged upon incubation at room temperature for 0, 4, 8, 24, and 48 h. The only change is the relative population of the two components, with the disordered component increasing slightly from ∼3.7% at 0 h to ∼5.5% at 48 h.

Intermolecular Distance Measurements in Aβ42 Globulomers To measure interspin distances using spin-spin interactions, we prepared globulomers using only spin-labeled Aβ42 (i.e. 100% labeled). Fig. 5A shows that the spectral amplitude of the 100% labeled sample (red traces) is reduced when compared with the 25% labeled sample (black traces), suggesting the existence of dipolar interactions. It should be noted that the EPR spectral line shape of the 100% labeled Aβ oligomer is dramatically different from the 100% labeled Aβ fibrils (28). In the parallel in-register β-structure of Aβ fibrils, spin labels are stacked on top of each other, giving rise to a single-line spectrum characteristic of strong spin exchange interactions (28). Clearly, the EPR spectra of the 100% labeled oligomers are indicative of dipolar interactions and do not have the single-line feature. Therefore, EPR data suggest that Aβ42 globulomers adopt structures different from fibrils. View larger version: Download as PowerPoint Slide FIGURE 5. Intermolecular distance measurements for spin-labeled Aβ42 globulomers. A, EPR spectra of 100% labeled oligomers at room temperature show significantly reduced spectral amplitude than 25% labeled samples, suggesting intermolecular dipolar interactions. B, EPR spectra of 25 and 100% labeled Aβ42 globulomers at 170 K. Interspin distances are obtained by simulating the 100% labeled spectra. The residual is the difference between simulated spectra and 100% labeled spectra. C, plot of intermolecular distance between spin labels and the percentage of spin labels at measured distances. D, plot of the populations of spin labels at >20 Å. Error bars in C and D indicate fitting errors. We measured intermolecular distances in Aβ42 globulomers using continuous wave EPR, which is sensitive to distances in the range of 8–20 Å (41). For distance measurements, EPR spectra were collected at 170 K. The EPR spectra are shown in Fig. 5B, and the measured distances are shown in Fig. 5C (top panel). The segment with the shortest distances is located at residues Gly-29–Val-40, with distances of 11.5–12.5 Å. Residues Phe-4–Ser-26 have slightly longer distances in the range of 12.5–13.5 Å. The two terminal residues Asp-1 and Ala-42 give distances >13.5 Å. The intermolecular distances from EPR provide structural restraints for the intermolecular organization of Aβ42 subunits in globulomers. First, Aβ42 globulomers consist of a tightly packed C-terminal region (Gly-29–Val-40) and a loosely packed N-terminal region. Second, the shortest distances observed at residues Gly-29–Val-40 are in the range of 11.5–12.5 Å, suggesting the absence of parallel in-register β-structures. Third, the intermolecular distances for most labeling positions are in a narrow range of 11.5–13.5 Å, suggesting an overall parallel arrangement for the Aβ42 subunits at this spacing. In Fig. 6, we depict four schematic models for the potential arrangement for the C-terminal region (Gly-29–Val-40). In Fig. 6, A and C, the measured interspin distances correspond to spacing between alternating β-strands within the same β-sheet. In Fig. 6, B and D, the interspin distances correspond to spacing between two face-to-back packed β-sheets. View larger version: Download as PowerPoint Slide FIGURE 6. Potential structural origins of measured intermolecular distances for C-terminal residues Gly-29–Ala-42. A–D, different β-strand organizations giving rise to the measured distances at ∼10 Å. Red balls represent spin labels. Numbers in A and C show approximate residue positions for each β-strand segment. Each model consists of only a minimum repeating unit, which can be extended in either hydrogen-bonding or side chain directions. From distance analysis, we obtained not only distances in the range of 8–20 Å, but also the population of spin labels at the measured distances. We also obtained population of spin labels at distances >20 Å, which do not contribute to spectral broadening. Fig. 5C (bottom panel) shows that only ∼25–50% of spin labels give rise to the measured distances in the range of 11.5–14.5 Å. The majority of the spin labels are at distances >20 Å (Fig. 5D). This information may also be useful in detailed structural modeling studies. For example, in Fig. 6A, spin labels on the green β-strand would fall into the category of >20 Å.