Out-of-Register Parallel ß-Sheets and Antiparallel ß-Sheets Coexist in 150-kDa Oligomers Formed by Amyloid-ß(1-42).

We present solid-state NMR measurements of ß-strand secondary structure and inter-strand organization within a 150-kDa oligomeric aggregate of the 42-residue variant of the Alzheimer's amyloid-ß peptide (Aß(1-42)). We build upon our previous report of a ß-strand spanned by residues 30-42, which arranges into an antiparallel ß-sheet. New results presented here indicate that there is a second ß-strand formed by residues 11-24. Contrary to expectations, NMR data indicate that this second ß-strand is organized into a parallel ß-sheet despite the co-existence of an antiparallel ß-sheet in the same structure. In addition, the in-register parallel ß-sheet commonly observed for amyloid fibril structure does not apply to residues 11-24 in the 150-kDa oligomer. Rather, we present evidence for an inter-strand registry shift of three residues that likely alternate in direction between adjacent molecules along the ß-sheet. We corroborated this unexpected scheme for ß-strand organization using multiple two-dimensional NMR and 13C-13C dipolar recoupling experiments. Our findings indicate a previously unknown assembly pathway and inspire a suggestion as to why this aggregate does not grow to larger sizes.

Solid-State NMR Structural Characterization of Self-Assembled Peptides with Selective 13C and 15N Isotopic Labels.

For the structural characterization methods discussed here, information on molecular conformation and intermolecular organization within nanostructured peptide assemblies is discerned through analysis of solid-state NMR spectral features. This chapter reviews general NMR methodologies, requirements for sample preparation, and specific descriptions of key experiments. An attempt is made to explain choices of solid-state NMR experiments and interpretation of results in a way that is approachable to a nonspecialist. Measurements are designed to determine precise NMR peak positions and line widths, which are correlated with secondary structures, and probe nuclear spin-spin interactions that report on three-dimensional organization of atoms. The formulation of molecular structural models requires rationalization of data sets obtained from multiple NMR experiments on samples with carefully chosen 13C and 15N isotopic labels. The information content of solid-state NMR data has been illustrated mostly through the use of simulated data sets and references to recent structural work on amyloid fibril-forming peptides and designer self-assembling peptides.

Antiparallel ß-Sheet Structure within the C-Terminal Region of 42-Residue Alzheimer's Amyloid-ß Peptides When They Form 150-kDa Oligomers.

Understanding the molecular structures of amyloid-ß (Aß) oligomers and underlying assembly pathways will advance our understanding of Alzheimer's disease (AD) at the molecular level. This understanding could contribute to disease prevention, diagnosis, and treatment strategies, as oligomers play a central role in AD pathology. We have recently presented a procedure for production of 150-kDa oligomeric samples of Aß(1-42) (the 42-residue variant of the Aß peptide) that are compatible with solid-state nuclear magnetic resonance (NMR) analysis, and we have shown that these oligomers and amyloid fibrils differ in intermolecular arrangement of ß-strands. Here we report new solid-state NMR constraints that indicate antiparallel intermolecular alignment of ß-strands within the oligomers. Specifically, 150-kDa Aß(1-42) oligomers with uniform (13)C and (15)N isotopic labels at I32, M35, G37, and V40 exhibit ß-strand secondary chemical shifts in 2-dimensional (2D) finite-pulse radiofrequency-driven recoupling NMR spectra, spatial proximities between I32 and V40 as well as between M35 and G37 in 2D dipolar-assisted rotational resonance spectra, and close proximity between M35 H(α) and G37 H(α) in 2D CHHC spectra. Furthermore, 2D dipolar-assisted rotational resonance analysis of an oligomer sample prepared with 30% labeled peptide indicates that the I32-V40 and M35-G37 contacts are between residues on different molecules. We employ molecular modeling to compare the newly derived experimental constraints with previously proposed geometries for arrangement of Aß molecules into oligomers.

Asymmetric biodegradable microdevices for cell-borne drug delivery.

Use of live cells as carriers for drug-laden particulate structures possesses unique advantages for drug delivery. In this work, we report on the development of a novel type of particulate structures called microdevices for cell-borne drug delivery. The microdevices were fabricated by soft lithography with a disklike shape. Each microdevice was composed of a layer of biodegradable thermoplastic such as poly(lactic-co-glycolic acid). One face of the thermoplastic layer was covalently grafted with a cell-adhesive polyelectrolyte such as poly-l-lysine. This asymmetric structure allowed the microdevices to bind to live cells through bulk mixing without causing cell aggregation. Moreover, the cell-microdevice complexes were largely stable, and the viability and proliferation ability of the cells were not affected by the microdevices over a week. In addition, sustained release of a mock drug from the microdevices was demonstrated. This type of microdevice promises to be clinically useful for sustained intravascular drug delivery.

The Alzheimer's amyloid-ß(1-42) peptide forms off-pathway oligomers and fibrils that are distinguished structurally by intermolecular organization.

Increasing evidence suggests that soluble aggregates of amyloid-ß (Aß) initiate the neurotoxicity that eventually leads to dementia in Alzheimer's disease. Knowledge on soluble aggregate structures will enhance our understanding of the relationship between structures and toxicities. Our group has reported a stable and homogeneous preparation of Aß(1-42) oligomers that has been characterized by various biophysical techniques. Here, we have further analyzed this species by solid state nuclear magnetic resonance (NMR) spectroscopy and compared NMR results to similar observations on amyloid fibrils. NMR experiments on Aß(1-42) oligomers reveal chemical shifts of labeled residues that are indicative of ß-strand secondary structure. Results from two-dimensional dipolar-assisted rotational resonance experiments indicate proximities between I31 aliphatic and F19 aromatic carbons. An isotope dilution experiment further indicates that these contacts between F19 and I31 are intermolecular, contrary to models of Aß oligomers proposed previously by others. For Aß(1-42) fibrils, we observed similar NMR lineshapes and inter-side-chain contacts, indicating similar secondary and quaternary structures. The most prominent structural differences between Aß(1-42) oligomers and fibrils were observed through measurements of intermolecular (13)C-(13)C dipolar couplings observed in PITHIRDS-CT experiments. PITHIRDS-CT data indicate that, unlike fibrils, oligomers are not characterized by in-register parallel ß-sheets. Structural similarities and differences between Aß(1-42) oligomers and fibrils suggest that folded ß-strand peptide conformations form early in the course of self-assembly and that oligomers and fibrils differ primarily in schemes of intermolecular organization. Distinct intermolecular arrangements between Aß(1-42) oligomers and fibrils may explain why this oligomeric state appears off-pathway for monomer self-assembly to fibrils.