Structural Consequences of Introducing Bioactive Domains to Designer ß-Sheet Peptide Self-Assemblies.

We applied solid- and solution-state nuclear magnetic resonance spectroscopy to examine the structure of multidomain peptides composed of self-assembling ß-sheet domains linked to bioactive domains. Bioactive domains can be selected to stimulate specific biological responses (e.g., via receptor binding), while the ß-sheets provide the desirable nanoscale properties. Although previous work has established the efficacy of multidomain peptides, molecular-level characterization is lacking. The bioactive domains are intended to remain solvent-accessible without being incorporated into the ß-sheet structure. We tested for three possible anticipated molecular-level consequences of introducing bioactive domains to ß-sheet-forming peptides: (1) the bioactive domain has no effect on the self-assembling peptide structure; (2) the bioactive domain is incorporated into the ß-sheet nanofiber; and (3) the bioactive domain interferes with self-assembly such that nanofibers are not formed. The peptides involved in this study incorporated self-assembling domains based on the (SL)6 motif and bioactive domains including a VEGF-A mimic (QK), an IGF-mimic (IGF-1c), and a de novo SARS-CoV-2 binding peptide (SBP3). We observed all three of the anticipated outcomes from our examination of peptides, illustrating the unintended structural effects that could adversely affect the desired biofunctionality and biomaterial properties of the resulting peptide hydrogel. This work is the first attempt to evaluate the structural effects of incorporating bioactive domains into a set of peptides unified by a similar self-assembling peptide domain. These structural insights reveal unmet challenges in the design of highly tunable bioactive self-assembling peptide hydrogels.

Distinct neurotoxic TDP-43 fibril polymorphs are generated by heterotypic interactions with α-Synuclein.

Amyloid aggregates of specific proteins constitute important pathological hallmarks in many neurodegenerative diseases, defining neuronal degeneration and disease onset. Recently, increasing numbers of patients show comorbidities and overlaps between multiple neurodegenerative diseases, presenting distinct phenotypes. Such overlaps are often accompanied by colocalizations of more than one amyloid protein, prompting the question of whether direct interactions between different amyloid proteins could generate heterotypic amyloids. To answer this question, we investigated the effect of α-synuclein (αS) on the DNA-binding protein TDP-43 aggregation inspired by their coexistence in pathologies such as Lewy body dementia and limbic predominant age-related TDP-43 encephalopathy. We previously showed αS and prion-like C-terminal domain (PrLD) of TDP-43 synergistically interact to generate toxic heterotypic aggregates. Here, we extend these studies to investigate whether αS induces structurally and functionally distinct polymorphs of PrLD aggregates. Using αS-PrLD heterotypic aggregates generated in two different stoichiometric proportions, we show αS can affect PrLD fibril forms. PrLD fibrils show distinctive residue level signatures determined by solid state NMR, dye-binding capability, proteinase K (PK) stability, and thermal stability toward SDS denaturation. Furthremore, by gold nanoparticle labeling and transmission electron microscopy, we show the presence of both αS and PrLD proteins within the same fibrils, confirming the existence of heterotypic amyloid fibrils. We also observe αS and PrLD colocalize in the cytosol of neuroblastoma cells and show that the heterotypic PrLD fibrils selectively induce synaptic dysfunction in primary neurons. These findings establish the existence of heterotypic amyloid and provide a molecular basis for the observed overlap between synucleinopathies and TDP-43 proteinopathies.

Thermochromic Behavior of Polydiacetylene Nanomaterials Driven by Charged Peptide Amphiphiles.

The tunability of chromatic phases adapted by chromogenic polymers such as polydiacetylene (PDA) is key to their utility for robust sensing applications. Here, we investigated the influence of charged peptide interactions on the structure-dependent thermochromicity of amphiphilic PDAs. Solid-state NMR and circular dichroism analyses show that our oppositely charged peptide-PDA samples have distinct degrees of structural order, with the coassembled sample being in between the ß-sheet-like positive peptide-PDA and the relatively disordered negative peptide-PDA. All solutions exhibit thermochromicity between 20 and 80 °C, whereby the hysteresis of the blue, planar phase is much larger than that of the red, twisted phase. Resonance Raman spectroscopy of films demonstrates that only coassemblies with electrostatic complementarity stabilize coexisting blue and red PDA phases. This work reveals the nature of the structural changes responsible for the thermally responsive chromatic transitions of biomolecule-functionalized polymeric materials and how this process can be directed by sequence-dictated electrostatic interactions.

Anatomy of a selectively coassembled ß-sheet peptide nanofiber.

Peptide self-assembly, wherein molecule A associates with other A molecules to form fibrillar ß-sheet structures, is common in nature and widely used to fabricate synthetic biomaterials. Selective coassembly of peptide pairs A and B with complementary partial charges is gaining interest due to its potential for expanding the form and function of biomaterials that can be realized. It has been hypothesized that charge-complementary peptides organize into alternating ABAB-type arrangements within assembled ß-sheets, but no direct molecular-level evidence exists to support this interpretation. We report a computational and experimental approach to characterize molecular-level organization of the established peptide pair, CATCH. Discontinuous molecular dynamics simulations predict that CATCH(+) and CATCH(-) peptides coassemble but do not self-assemble. Two-layer ß-sheet amyloid structures predominate, but off-pathway ß-barrel oligomers are also predicted. At low concentration, transmission electron microscopy and dynamic light scattering identified nonfibrillar ∼20-nm oligomers, while at high concentrations elongated fibers predominated. Thioflavin T fluorimetry estimates rapid and near-stoichiometric coassembly of CATCH(+) and CATCH(-) at concentrations ≥100 µM. Natural abundance 13C NMR and isotope-edited Fourier transform infrared spectroscopy indicate that CATCH(+) and CATCH(-) coassemble into two-component nanofibers instead of self-sorting. However, 13C-13C dipolar recoupling solid-state NMR measurements also identify nonnegligible AA and BB interactions among a majority of AB pairs. Collectively, these results demonstrate that strictly alternating arrangements of ß-strands predominate in coassembled CATCH structures, but deviations from perfect alternation occur. Off-pathway ß-barrel oligomers are also suggested to occur in coassembled ß-strand peptide systems.

Side-Chain Chemistry Governs Hierarchical Order of Charge-Complementary ß-sheet Peptide Coassemblies.

Self-assembly of proteinaceous biomolecules into functional materials with ordered structures that span length scales is common in nature yet remains a challenge with designer peptides under ambient conditions. This report demonstrates how charged side-chain chemistry affects the hierarchical co-assembly of a family of charge-complementary ß-sheet-forming peptide pairs known as CATCH(X+/Y-) at physiologic pH and ionic strength in water. In a concentration-dependent manner, the CATCH(6K+) (Ac-KQKFKFKFKQK-Am) and CATCH(6D-) (Ac-DQDFDFDFDQD-Am) pair formed either ß-sheet-rich microspheres or ß-sheet-rich gels with a micron-scale plate-like morphology, which were not observed with other CATCH(X+/Y-) pairs. This hierarchical order was disrupted by replacing D with E, which increased fibril twisting. Replacing K with R, or mutating the N- and C-terminal amino acids in CATCH(6K+) and CATCH(6D-) to Qs, increased observed co-assembly kinetics, which also disrupted hierarchical order. Due to the ambient assembly conditions, active CATCH(6K+)-green fluorescent protein fusions could be incorporated into the ß-sheet plates and microspheres formed by the CATCH(6K+/6D-) pair, demonstrating the potential to endow functionality.

Oligomer Formation by Amyloid-ß42 in a Membrane-Mimicking Environment in Alzheimer's Disease.

The brains of Alzheimer's disease (AD) patients contain numerous amyloid plaques that are diagnostic of the disease. The plaques are primarily composed of the amyloidogenic peptides proteins Aß40 and Aß42, which are derived by the processing of the amyloid pre-cursor protein (APP) by two proteases called ß-secretase and γ-secretase. Aß42 differs from Aß40 in having two additional hydrophobic amino acids, ILE and ALA, at the C-terminus. A small percentage of AD is autosomal dominant (ADAD) and linked either to the genes for the presenilins, which are part of γ-secretase, or APP. Because ADAD shares most pathogenic features with widespread late-onset AD, Aß peptides have become the focus of AD research. Fibrils formed by the aggregation of these peptides are the major component of plaques and were initially targeted in AD therapy. However, the fact that the abundance of plaques does not correlate well with cognitive decline in AD patients has led investigators to examine smaller Aß aggregates called oligomers. The low levels and heterogeneity of Aß oligomers have made the determination of their structures difficult, but recent structure determinations of oligomers either formed or initiated in detergents have been achieved. We report here on the structures of these oligomers and suggest how they may be involved in AD.

Conformations of peptoids in nanosheets result from the interplay of backbone energetics and intermolecular interactions.

The conformations adopted by the molecular constituents of a supramolecular assembly influence its large-scale order. At the same time, the interactions made in assemblies by molecules can influence their conformations. Here we study this interplay in extended flat nanosheets made from nonnatural sequence-specific peptoid polymers. Nanosheets exist because individual polymers can be linear and untwisted, by virtue of polymer backbone elements adopting alternating rotational states whose twists oppose and cancel. Using molecular dynamics and quantum mechanical simulations, together with experimental data, we explore the design space of flat nanostructures built from peptoids. We show that several sets of peptoid backbone conformations are consistent with their being linear, but the specific combination observed in experiment is determined by a combination of backbone energetics and the interactions made within the nanosheet. Our results provide a molecular model of the peptoid nanosheet consistent with all available experimental data and show that its structure results from a combination of intra- and intermolecular interactions.

Biomolecular Assemblies: Moving from Observation to Predictive Design.

Biomolecular assembly is a key driving force in nearly all life processes, providing structure, information storage, and communication within cells and at the whole organism level. These assembly processes rely on precise interactions between functional groups on nucleic acids, proteins, carbohydrates, and small molecules, and can be fine-tuned to span a range of time, length, and complexity scales. Recognizing the power of these motifs, researchers have sought to emulate and engineer biomolecular assemblies in the laboratory, with goals ranging from modulating cellular function to the creation of new polymeric materials. In most cases, engineering efforts are inspired or informed by understanding the structure and properties of naturally occurring assemblies, which has in turn fueled the development of predictive models that enable computational design of novel assemblies. This Review will focus on selected examples of protein assemblies, highlighting the story arc from initial discovery of an assembly, through initial engineering attempts, toward the ultimate goal of predictive design. The aim of this Review is to highlight areas where significant progress has been made, as well as to outline remaining challenges, as solving these challenges will be the key that unlocks the full power of biomolecules for advances in technology and medicine.

Post-assembly α-helix to ß-sheet structural transformation within SAF-p1/p2a peptide nanofibers.

We report an unanticipated helix-to-sheet structural transformation within an assembly of SAF-p1 and SAF-p2a designer peptides. Solid-state NMR spectroscopic data support the assembled structure that was targeted by rational peptide design: an α-helical coiled-coil co-assembly of both peptides. Subsequent to assembly, however, the system converts to a ß-sheet structure that continues to exhibit nearest-neighbor interactions between the two peptide components. The structural transition occurs at pH 7.4 and exhibits strongly temperature-dependent kinetics between room temperature (weeks) and 40 °C (minutes). We further observed evidence of reversibility on the timescale of months at 4 °C. The structural conversion from the anticipated structure to an unexpected structure highlights an important aspect to the challenge of designing peptide assemblies. Furthermore, the conformational switching mechanism mediated by a prerequisite α-helical nanostructure represents a previously unknown route for ß-sheet designer peptide assembly.

Assembly of short amphiphilic peptoids into nanohelices with controllable supramolecular chirality.

A long-standing challenge in bioinspired materials is to design and synthesize synthetic materials that mimic the sophisticated structures and functions of natural biomaterials, such as helical protein assemblies that are important in biological systems. Herein, we report the formation of a series of nanohelices from a type of well-developed protein-mimetics called peptoids. We demonstrate that nanohelix structures and supramolecular chirality can be well-controlled through the side-chain chemistry. Specifically, the ionic effects on peptoids from varying the polar side-chain groups result in the formation of either single helical fiber or hierarchically stacked helical bundles. We also demonstrate that the supramolecular chirality of assembled peptoid helices can be controlled by modifying assembling peptoids with a single chiral amino acid side chain. Computational simulations and theoretical modeling predict that minimizing exposure of hydrophobic domains within a twisted helical form presents the most thermodynamically favorable packing of these amphiphilic peptoids and suggests a key role for both polar and hydrophobic domains on nanohelix formation. Our findings establish a platform to design and synthesize chiral functional materials using sequence-defined synthetic polymers.

Parallel ß-Sheet Structure and Structural Heterogeneity Detected within Q11 Self-Assembling Peptide Nanofibers.

Q11 peptide nanofibers are used as a biomaterial for applications such as antigen presentation and tissue engineering, yet detailed knowledge of molecular-level structure has not been reported. The Q11 peptide sequence was designed using heuristics-based patterning of hydrophobic and polar amino acids with oppositely charged amino acids placed at opposite ends of the sequence to promote antiparallel ß-sheet formation. In this work, we employed solid-state nuclear magnetic resonance spectroscopy (NMR) to evaluate whether the molecular organization within Q11 self-assembled peptide nanofibers is consistent with the expectations of the peptide designers. We discovered that Q11 forms a distribution of molecular structures. NMR data from two-dimensional (2D) 13C-13C dipolar-assisted rotational resonance indicate that the K3 and E9 residues between Q11 ß-strands are spatially proximate (within ∼0.6 nm). Frequency-selective rotational echo double resonance (fsREDOR) on K3 Nζ and E9 Cδ-labeled sites showed that approximately 9% of the sites are close enough for salt bridge formation to occur. Surprisingly, dipolar recoupling measurements revealed that Q11 peptides do not assemble into antiparallel ß-sheets as expected, and structural analysis using Fourier-transform infrared spectroscopy and 2D NMR alone can be misleading. 13C PITHIRDS-CT dipolar recoupling measurements showed that the most abundant structure consists of parallel ß-sheets, in contrast to the expected antiparallel ß-sheet structure. Structural heterogeneity was detected from 15N{13C} REDOR measurements, with approximately 22% of ß-strands having antiparallel nearest neighbors. We cannot propose a complete structural model of Q11 nanofibers because of the complexity involved when examining structurally heterogeneous samples using NMR. Altogether, our results show that while heuristics-based patterning is effective in promoting ß-sheet formation, designing a peptide sequence to form a targeted ß-strand arrangement remains challenging.

Investigation of Rare Earth Element Binding to a Surface-Bound Affinity Peptide Derived from EF-Hand Loop I of Lanmodulin.

Bioinspired strategies have been given extensive attention for the recovery of rare earth elements (REEs) from waste streams because of their high selectivity, regeneration potential, and sustainability as well as low cost. Lanmodulin protein is an emerging biotechnology that is highly selective for REE binding. Mimicking lanmodulin with shorter peptides is advantageous because they are simpler and potentially easier to manipulate and optimize. Lanmodulin-derived peptides have been found to bind REEs, but their properties have not been explored when immobilized on solid substrates, which is required for many advanced separation technologies. Here, two peptides, LanM1 and scrambled LanM1, are designed from the EF-hand loop 1 of lanmodulin and investigated for their binding affinity toward different REEs when surface-bound. First, the ability of LanM1 to bind REEs was confirmed and characterized in solution using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations for Ce(III) ions. Isothermal titration calorimetry (ITC) was used to further analyze the binding of the LanM1 to Ce(III), Nd(III), Eu(III), and Y(III) ions and in low-pH conditions. The performance of the immobilized peptides on a model gold surface was examined using a quartz crystal microbalance with dissipation (QCM-D). The studies show that the LanM1 peptide has a stronger REE binding affinity than that of scrambled LanM1 when in solution and when immobilized on a gold surface. QCM-D data were fit to the Langmuir adsorption model to estimate the surface-bound dissociation constant (Kd) of LanM1 with Ce(III) and Nd(III). The results indicate that LanM1 peptides maintain a high affinity for REEs when immobilized, and surface-bound LanM1 has no affinity for potential competitor calcium and copper ions. The utility of surface-bound LanM1 peptides was further demonstrated by immobilizing them to gold nanoparticles (GNPs) and capturing REEs from solution in experiments utilizing an Arsenazo III-based colorimetric dye displacement assay and ultraviolet-visible (UV-vis) spectrophotometry. The saturated adsorption capacity of GNPs was estimated to be around 3.5 µmol REE/g for Ce(III), Nd(III), Eu(III), and Y(III) ions, with no binding of non-REE Ca(II) ions observed.

Self-Assembling Peptides with Insulin-Like Growth Factor Mimicry.

Growth factor (GF) mimicry involves recapitulating the signaling of larger molecules or cells. Although GF mimicry holds considerable promise in tissue engineering and drug design applications, difficulties in targeting the signaling molecule to the site of delivery and dissociation of mimicking peptides from their target receptors continue to limit its clinical application. To address these challenges, we utilized a self-assembling peptide (SAP) platform to generate synthetic insulin-like growth factor (IGF)-signaling, self-assembling GFs. Our peptide hydrogels are biocompatible and bind target IGF receptors in a dose-dependent fashion, activate proangiogenic signaling, and facilitate formation of angiogenic microtubules in vitro. Furthermore, infiltrated hydrogels are stable for weeks to months. We conclude that the enhanced targeting and long-term stability of our SAP/GF mimicry implants may improve the efficacy and safety of future GF mimic therapeutics.

Antiviral fibrils of self-assembled peptides with tunable compositions.

The lasting threat of viral pandemics necessitates the development of tailorable first-response antivirals with specific but adaptive architectures for treatment of novel viral infections. Here, such an antiviral platform has been developed based on a mixture of hetero-peptides self-assembled into functionalized ß-sheets capable of specific multivalent binding to viral protein complexes. One domain of each hetero-peptide is designed to specifically bind to certain viral proteins, while another domain self-assembles into fibrils with epitope binding characteristics determined by the types of peptides and their molar fractions. The self-assembled fibrils maintain enhanced binding to viral protein complexes and retain high resilience to viral mutations. This method is experimentally and computationally tested using short peptides that specifically bind to Spike proteins of SARS-CoV-2. This platform is efficacious, inexpensive, and stable with excellent tolerability.

Solid-state NMR evidence for ß-hairpin structure within MAX8 designer peptide nanofibers.

MAX8, a designer peptide known to undergo self-assembly following changes in temperature, pH, and ionic strength, has demonstrated usefulness for tissue engineering and drug delivery. It is hypothesized that the self-assembled MAX8 nanofiber structure consists of closed ß-hairpins aligned into antiparallel ß-sheets. Here, we report evidence from solid-state NMR spectroscopy that supports the presence of the hypothesized ß-hairpin conformation within the nanofiber structure. Specifically, our (13)C-(13)C two-dimensional exchange data indicate spatial proximity between V3 and K17, and (13)C-(13)C dipolar coupling measurements reveal proximity between the V3 and V18 backbone carbonyls. Moreover, isotopic dilution of labeled MAX8 nanofibers did not result in a loss of the (13)C-(13)C dipolar couplings, showing that these couplings are primarily intramolecular. NMR spectra also indicate the existence of a minor conformation, which is discussed in terms of previously hypothesized nanofiber physical cross-linking and possible nanofiber polymorphism.

Traffic jam at the blood-brain barrier promotes greater accumulation of Alzheimer's disease amyloid-ß proteins in the cerebral vasculature.

Amyloid-ß (Aß) deposition in the brain vasculature results in cerebral amyloid angiopathy (CAA), which occurs in about 80% of Alzheimer's disease (AD) patients. While Aß42 predominates parenchymal amyloid plaques in AD brain, Aß40 is prevalent in the cerebrovascular amyloid. Dutch mutation of Aß40 (E22Q) promotes aggressive cerebrovascular accumulation and leads to severe CAA in the mutation carriers; knowledge of how DutchAß40 drives this process more efficiently than Aß40 could reveal various pathophysiological events that promote CAA. In this study we have demonstrated that DutchAß40 shows preferential accumulation in the blood-brain-barrier (BBB) endothelial cells due to its inefficient blood-to-brain transcytosis. Consequently, DutchAß40 establishes a permeation barrier in the BBB endothelium, prevents its own clearance from the brain, and promotes the formation of amyloid deposits in the cerebral microvessels. The BBB endothelial accumulation of native Aß40 is not robust enough to exercise such a significant impact on its brain clearance. Hence, the cerebrovascular accumulation of Aß40 is slow and may require other copathologies to precipitate into CAA. In conclusion, the magnitude of Aß accumulation in the BBB endothelial cells is a critical factor that promotes CAA; hence, clearing vascular endothelium of Aß proteins may halt or even reverse CAA.

Distinct solid and solution state self-assembly pathways of RADA16-I designer peptide.

Solid state NMR measurements on selectively (13) C-labeled RADA16-I peptide (COCH3 -RADARADARADARADA-NH2 ) were used to obtain new molecular level information on the conversion of α-helices to ß-sheets through self-assembly in the solid state with increasing temperature. Isotopic labeling at the A4 Cß site enabled rapid detection of (13) C NMR signals. Heating to 344-363 K with simultaneous NMR detection allowed production of samples with systematic variation of α-helix and ß-strand content. These samples were then probed at room temperature for intermolecular (13) C-(13) C nuclear dipolar couplings with the PITHIRDS-CT NMR experiment. The structural transition was also characterized by Fourier transform infrared spectroscopy and wide angle X-ray diffraction. Independence of PITHIRDS-CT decay shapes on overall α-helical and ß-strand content infers that ß-strands are not observed without association with ß-sheets, indicating that ß-sheets are formed at elevated temperatures on a timescale that is fast relative to the NMR experiment. PITHIRDS-CT NMR data were compared with results of similar measurements on RADA16-I nanofibers produced by self-assembly in aqueous salt solution. We report that ß-sheets formed through self-assembly in the solid state have a structure that differs from those formed through self-assembly in the solution state. Specifically, solid state RADA16-I self-assembly produces in-register parallel ß-sheets, whereas nanofibers are composed of stacked parallel ß-sheets with registry shifts between adjacent ß-strands in each ß-sheet. These results provide evidence for environment-dependent self-assembly mechanisms for RADA16-I ß-sheets as well as new constraints on solid state self-assembled structures, which must be avoided to maximize solution solubility and nanofiber yields.

A common pathway for detergent-assisted oligomerization of Aß42.

Amyloid beta (Aß) aggregation is a slow process without seeding or assisted nucleation. Sodium dodecyl sulfate (SDS) micelles stabilize Aß42 small oligomers (in the dimer to tetramer range); subsequent SDS removal leads to a 150-kD Aß42 oligomer. Dodecylphosphorylcholine (DPC) micelles also stabilize an Aß42 tetramer. Here we investigate the detergent-assisted oligomerization pathway by solid-state NMR spectroscopy and molecular dynamics simulations. SDS- and DPC-induced oligomers have the same structure, implying a common oligomerization pathway. An antiparallel ß-sheet formed by the C-terminal region, the only stable structure in SDS and DPC micelles, is directly incorporated into the 150-kD oligomer. Three Gly residues (at positions 33, 37, and 38) create holes that are filled by the SDS and DPC hydrocarbon tails, thereby turning a potentially destabilizing feature into a stabilizing factor. These observations have implications for endogenous Aß aggregation at cellular interfaces.

Solid state self-assembly mechanism of RADA16-I designer peptide.

We report that synthetic RADA16-I peptide transforms to ß-strand secondary structure and develops intermolecular organization into ß-sheets when stored in the solid state at room temperature. Secondary structural changes were probed using solid state nuclear magnetic resonance spectroscopy (ssNMR) and Fourier transform infrared spectroscopy (FTIR). Intermolecular organization was analyzed via wide-angle X-ray diffraction (WAXD). Observed changes in molecular structure and organization occurred on the time scale of weeks during sample storage at room temperature. We observed structural changes on faster time scales by heating samples above room temperature or by addition of water. Analysis of hydration effects indicates that water can enhance the ability of the peptide to convert to ß-strand secondary structure and assemble into ß-sheets. However, temperature dependent FTIR and time dependent WAXD data indicate that bound water may hinder the assembly of ß-strands into ß-sheets. We suggest that secondary structural transformation and intermolecular organization together produce a water-insoluble state. These results reveal insights into the role of water in self-assembly of polypeptides with hydrophilic side chains, and have implications on future optimization of RADA16-I nanofiber production.

Seeded growth of beta-amyloid fibrils from Alzheimer's brain-derived fibrils produces a distinct fibril structure.

Studies by solid-state nuclear magnetic resonance (NMR) of amyloid fibrils prepared in vitro from synthetic 40-residue beta-amyloid (Abeta(1-40)) peptides have shown that the molecular structure of Abeta(1-40) fibrils is not uniquely determined by amino acid sequence. Instead, the fibril structure depends on the precise details of growth conditions. The molecular structures of beta-amyloid fibrils that develop in Alzheimer's disease (AD) are therefore uncertain. We demonstrate through thioflavin T fluorescence and electron microscopy that fibrils extracted from brain tissue of deceased AD patients can be used to seed the growth of synthetic Abeta(1-40) fibrils, allowing preparation of fibrils with isotopic labeling and in sufficient quantities for solid-state NMR and other measurements. Because amyloid structures propagate themselves in seeded growth, as shown in previous studies, the molecular structures of brain-seeded synthetic Abeta(1-40) fibrils most likely reflect structures that are present in AD brain. Solid-state (13)C NMR spectra of fibril samples seeded with brain material from two AD patients were found to be nearly identical, indicating the same molecular structures. Spectra of an unseeded control sample indicate greater structural heterogeneity. (13)C chemical shifts and other NMR data indicate that the predominant molecular structure in brain-seeded fibrils differs from the structures of purely synthetic Abeta(1-40) fibrils that have been characterized in detail previously. These results demonstrate a new approach to detailed structural characterization of amyloid fibrils that develop in human tissue, and to investigations of possible correlations between fibril structure and the degree of cognitive impairment and neurodegeneration in AD.