(B) Ribbon representation from the cylindrin crystal structure. a = 4): **, 0.01; and ***, 0.001. Table 1 Structures and stoichiometries of amyloid-related oligomers discussed in this report, derived from alphaB crystallin. thead th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Protein/Peptide Segment (residue numbers)a /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Structure /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ PDB ID /th th colspan=”3″ align=”center” valign=”top” rowspan=”1″ Oligomer Size by /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Immunoreactivity /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Toxicity /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Crystallography /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ SEC /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ PF-6260933 MS /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ /th /thead alphaB crystallin truncated (68C162)Network of dimers linked by domain swapping3L1G2 to indefinitely large2C4b2C6 & 10C24b-NocK11VCylindrin3SGO6-6d–K11VV2LCylindrin V2L variant3SGP666eA11YesK11V-TRCylindrin tandem repeat V2L variant3SGR333eA11YesK11VV4W-TRCylindrin tandem repeat V2L/V4W variant–1C2-NofNoG6VFibril steric zipper3SGSIndefinitely large–NoNo Open in a separate window aPeptide segment amino acid sequences are provided in Table SI. bData refer to truncated alphaB crystallin (16). cData refer PF-6260933 to full length alphaB crystallin (50). dData not shown. eData shown in Fig S3. fVery weak binding. Data shown in Fig. S1E. C, Sample was not tested or unknown. SEC, Size exclusion chromatography. MS, Native mass spectrometry. The hairpin, segment KVKVLGDVIEV (termed K11V) formed both amyloid fibrils and oligomers. Upon shaking at elevated temperature, K11V forms fibrils similar to those of the parent protein (ABC) from which the segment is derived (15) and similar to those of a tandem repeat of K11V (K11V-TR) (Fig. 1B, Fig. S1BCC, and TSPAN7 Table S1). The fibrils range from 20 to 100 nm in diameter as viewed by electron microscopy (Fig. S1). X-ray diffraction of dried fibrils displayed rings at 4.8 and 12 ? resolution, consistent with the signature cross-beta pattern of amyloid fibrils (Fig. S1C). The amyloid fibrils of K11V-TR bind the specific amyloid dye congo-red, producing apple-green bifringerance under polarized light (Fig. S1D), and are immunoreactive with the fibril-specific, conformation-dependent antibody, OC (Fig. 1E and Fig. S1E) (20). Together these results prove that the segments G6V, K11V, and K11V-TR are all capable of converting to the amyloid state (21, 22), as is their parent protein, ABC. Under physiological conditions, the segment K11V, K11V-TR, and a sequence variant with Leu replacing Val at position 2 (K11VV2L), all form stable small oligomers, intermediate in size between monomer and fiber. For each sequence, we determined the number of molecules in the oligomers by size exclusion chromatography (SEC-HPLC) and native mass spectrometry experiments. Purified recombinant K11VV2L, and K11V-TR, a tandem repeat of K11VV2L eluted as oligomeric complexes by SEC (Fig. 1C). For example, the K11V-TR complex was estimated to be ~8 kDa in mass, corresponding roughly to three tandem segment chains. As an additional check on the stoichiometry of the tandem repeat K11V-TR oligomer, we subjected peak fractions to native nanoelectrospray mass spectrometry. Mass spectra clearly showed abundant ions of K11V-TR oligomers with masses corresponding PF-6260933 to three peptide chains (Fig. 1D and S3). Furthermore, we were able to isolate ions of the K11V-TR oligomer and perform collision induced dissociation (CID) of this trimeric peptide complex into monomeric units of mass equal to the K11V-TR PF-6260933 peptide (Fig. PF-6260933 S4). Similar experiments show that K11V and K11VV2L form hexameric oligomers (Table 1 and Fig. S3). Thus native mass spectrometry is consistent with SEC-HPLC in suggesting a stoichiometry of a K11V oligomer of six chains and a K11V-TR oligomer of three tandem chains. These results are consistent with crystallography and energetic considerations (see below). These ABC K11V oligomers exhibit molecular properties in common with amyloid oligomers from other disease-related proteins. We probed blots of the recombinant segments with the polyclonal A11, amyloid-oligomer-specific conformational antibody (5). Both single and tandem repeat segments are recognized by the A11 antibody (Fig. 1E and Fig. S1E). Using a cell viability assay on mammalian cells, we observed oligomers to be toxic, displaying dose-response effects similar to those of Abeta involved in Alzheimer’s disease (2, 23, 24) (Fig. 1F and Fig. S5). To test if membrane disruption is responsible for this toxicity, as suggested for human Islet Amyloid Polypeptide (hIAPP) (25, 26), we performed liposome dye-release experiments. The hIAPP peptide clearly diminished liposome integrity leading to.