MATERIALS AND METHODS RESULTS Computational Electronic structures for 20 full-length Aβ40 conformer structures and inter-atomic interaction energies from these electronic structures using natural bonding orbital (NBO) formalism revealed: 1. C-terminus:helix region interactions 2. N-terminus:helix region interactions 3. strong interactions of residues within the 15-24 region that stabilize the helix 4. interactions between residues 40-35, 33-27, and 3-9 Experimental Structure 3 synthesis 1. lactam bridge between Asp23 and a Lys residue that we substituted for Val40 2. covalent linkage prevents interaction of the C-terminus with the core helix 3. predicted to preclude formation of the stable dimer state formed through concerted collapse of two monomers and produce distinct assembly behavior [ENGINEERED PEPTIDES. Design principles and positions of lactam bridges () are noted above each structure. Predicted effects are shown to the right of each structure. ka is assembly rate. Peptide regions: green, N-terminal; red, central helical (His13–Asp23); blue, C-terminal.] SDS-PAGE showed that structure 3 vs Aβ40 1. displayed no dimer band, but an intense monomer band 2. a band migrating below monomer 3. suggesting that structure 3 is capable of forming two conformers in SDS-PAGE, one with an Mr consistent with wild type Aβ40 and one with a smaller Mr of ≈3500. [SDS-PAGE/SILVER STAINING OF Aβ40 AND STRUCTURE 3 AFTER 10 D INCUBATION. “MWM” signifies molecular weight markers. Arrows: _2_, dimer; _1_, monomer; _a_, band of Mr ≈ 3500.] Mass spectrometry of structure 3 vs Aβ40 revealed: 1. structure 3 spectrum contained a large -7/2 dimer (formed from -4 and -3 monomers) peak 2. suggesting that self association of structure 3 produces a dimer structure unique from that formed by wild type Aβ40 Ion mobility spectroscopy (IMS) studies of structure 3 vs Aβ40 revealed: 1. “collision cross section” (σ) value for structure 3 was larger than that of wild type Aβ40 (620 vs 607 Å²) 2. supporting the explanation that structure 3 forms a unique conformer [MASS SPECTROMETRY. Negative ion MS of wild type Aβ40 and Structure 3 (inset). Scale of abscissa is m/z, where m=mass and z=charge. Numbers above peaks are z/n, where n is number of monomers.] ThT fluorescence to monitor formation of extended β-sheet in structure 3 vs Aβ40 revealed: 1. an immediate monotonic increase in fluorescence that plateaued at 12 days at a level that was ≈1/2 that of Aβ40 2. appears to self-associate without a substantial lag phase (<<1 days) 3. appears to produce aggregates that display substantially less β-sheet than do wild type assemblies [THT FLUORESCENCE.] EM to compare assembly morphology of structure 3 vs Aβ40 revealed: 1. far fewer fibrils 2. a number of structures not seen with WT Aβ40, including short worm-like structures, longer worm-like structures, and fibrils with prominent helical twists 3. multifilar rod-like structures 4. some of the rod-like structures had “sausage-link” morphologies with the links occurring at intervals of ≈270 nm
Eric Y. Haydena, Kimberly K. Hoia#, Jasmine Lopeza, Mohammed Inayathullahc, Margaret M. Condrona, and David B. Teplowab* aDepartment of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 bMolecular Biology Institute and Brain Research Institutes; UCLA, Los Angeles, CA 90095 cBiomaterials and Advanced Drug Delivery Laboratory, School of Medicine, Stanford University, Palo Alto, CA 94304, USA #Current address: Department of Pediatrics and Department of Neurology, UCSF, San Francisco, CA 94158 *To whom correspondence should be addressed.