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Previous reports revealed that sodium dodecyl sulfate near its critical micelle concentration can drive the assembly of Aβ42 along an oligomeric pathway. This pathway produces a 150 kDa peptide oligomer (approximately 32 peptide molecules or protomers) that does not aggregate further into amyloid fibrils. Solid-state nuclear magnetic resonance (NMR) spectroscopy revealed structural features distinguishing the 150 kDa oligomer from fibrils. A puzzling feature was the coexistence of parallel and antiparallel β-sheets within the oligomer structure. Here we present new atomic-level structural constraints obtained via solid-state NMR spectroscopy, benefitting from improved resolution via sample concentration by ultracentrifugation. In addition, two-dimensional cryo-electron microscopy (cryo-EM) reconstruction revealed a 4-fold symmetric shape. We propose a structural model to rationalize the solid-sate NMR- and cryo-EM-derived structural constraints. This model has a hollow square cylinder shape, with antiparallel β-sheets formed by residues 33-39 lining the inner walls and parallel β-sheets formed by residues 11-22 lining the outer walls. Within successive layers, the outer β-strands on each side of the square cylinder alternate between two forms: one within a U-shaped protomer and another within L-shaped protomer. Molecular dynamics simulations show that, when the oligomer model is embedded in a lipid membrane, ions permeate through the central pore, with cation selectivity. The model further motivates an assembly pathway-based interpretation that may explain why the 150 kDa oligomer does not undergo further aggregation into amyloid fibrils.Significance StatementAβ oligomers are thought to be the most toxic species in Alzheimer’s disease. Their sizes range from 2 to ∼50 protomers. Most published experimental data on Aβ oligomers indicate that they, like fibrils, are composed of β-sheets, but it is a mystery why any β-sheet aggregate would exist as a stable oligomer without undergoing further aggregation into fibrils. Here, structural constraints from solid-state NMR and cryo-EM led us to an oligomer model with a hollow square cylinder shape capable of conducting ions when embedded in a lipid membrane. Based on the model, we argue that geometric frustration may distinguish the assembly pathway that produces this oligomer from fibril-forming assembly pathways.

Screening amino acid sequence space via experiments to discover peptides that self-assemble into amyloid fibrils is challenging. We have developed a computational peptide assembly design (PepAD) algorithm that enables the discovery of amyloid-forming peptides. Discontinuous molecular dynamics (DMD) simulation with the PRIME20 force field combined with the FoldAmyloid tool is used to examine the fibrilization kinetics of PepAD-generated peptides. PepAD screening of ∼10,000 7-mer peptides resulted in twelve top-scoring peptides with two distinct hydration properties. Our studies revealed that eight of the twelve in silico discovered peptides spontaneously form amyloid fibrils in the DMD simulations and that all eight have at least five residues that the FoldAmyloid tool classifies as being aggregation-prone. Based on these observations, we re-examined the PepAD-generated peptides in the sequence pool returned by PepAD and extracted five sequence patterns as well as associated sequence signatures for the 7-mer amyloid-forming peptides. Experimental results from Fourier transform infrared spectroscopy (FTIR), thioflavin T (ThT) fluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) indicate that all the peptides predicted to assemble in silico assemble into antiparallel β-sheet nanofibers in a concentration-dependent manner. This is the first attempt to use a computational approach to search for amyloid-forming peptides based on customized settings. Our efforts facilitate the identification of β-sheet-based self-assembling peptides, and contribute insights towards answering a fundamental scientific question: “What does it take, sequence-wise, for a peptide to self-assemble?”

Amyloid aggregates of specific proteins form important pathological hallmarks in many neurodegenerative diseases, defining neuronal degeneration and disease onset. Recently, increasing numbers of patients show co-morbidities and overlaps between multiple neurodegenerative diseases, presenting distinct phenotypes. Such overlaps are often accompanied by co-localizations 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 TDP-43 aggregation inspired by their co-existence in pathologies such as Lewy body dementia and limbic predominant age-related TDP-43 encephalopathy. We previously showed that αS and prion-like C-terminal domain (PrLD) of TDP-43 synergistically interact with one another to generate toxic heterotypic aggregates in vitro. 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 that αS can effect PrLD fibril forms. The fibril samples have distinctive residue-level structural signatures in NMR spectra, dye-binding capability, proteinase K (PK) stability, and SDS-sensitive thermal stability. By gold nanoparticle labeling and TEM, we show the presence of both αS and PrLD proteins within the same fibrils, and thus the existence of hetertypic hybrid fibrils. We also observe that αS and PrLD co-localize in the cytosol of SH-SY5Y neuroblastoma cells, and show that the heterotypic PrLD fibrils selectively induce synaptic dysfunction in primary cortical neurons. These findings establish the existence of heterotypic amyloid polymorphs and provide a molecular basis for the observed overlap between synucleinopathies and TDP-43 proteinopathies.

Coassembling peptides offer an additional degree of freedom in the design of nanostructured biomaterials when compared to analogous self-assembling peptides. Yet, our understanding of how amino acid sequences encodes coassembled nanofiber structure is limited. Prior work on a charge-complementary pair, CATCH+ and CATCH- peptides, detected like-peptide nearest neighbors (CATCH+:CATCH+ and CATCH-:CATCH-) within coassembled β-sheet nanofibers; these self-associated peptide pairs marked a departure from an "ideal" coassembled structure. In this work, we employ solid-state NMR, isotope-edited FTIR, and coarse-grained molecular dynamics simulations to evaluate the alignment of β-strands within CATCH peptide nanofibers. Both experimental and computational results suggest that CATCH molecules coassemble into structurally heterogeneous nanofibers, which is consistent with our observations in another coassembling system, the King-Webb peptides. Within β-sheet nanofibers, β-strands were found to have nearest neighbors aligned in-register parallel, in-register antiparallel, and out-of-register. In comparison to the King-Webb peptides, CATCH nanofibers exhibit a greater degree of structural heterogeneity. By comparing the amino acid sequences of CATCH and King-Webb peptides, we can begin to unravel sequence-to-structure relationships, which may encode more precise coassembled β-sheet nanostructures.

Peptide self-assembly, wherein molecule A associates with other A molecules to form fibrillar β-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 β-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 β-sheet amyloid structures predominate, but off-pathway β-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 13 C NMR and isotope-edited Fourier transform infrared spectroscopy indicate that CATCH(+) and CATCH(−) coassemble into two-component nanofibers instead of self-sorting. However, 13 C– 13 C 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 β-strands predominate in coassembled CATCH structures, but deviations from perfect alternation occur. Off-pathway β-barrel oligomers are also suggested to occur in coassembled β-strand peptide systems.

Self-assembling peptides have garnered an increasing amount of interest as a functional biomaterial for medical and biotechnological applications. Recently, β-sheet peptide designs utilizing complementary pairs of peptides composed of charged amino acids positioned to impart co-assembly behavior have expanded the portfolio of peptide aggregate structures. Structural characterization of these charge-complementary peptide co-assemblies has been limited. Thus, it is not known how the complementary peptides organize on the molecular level. Through a combination of solid-state NMR measurements and discontinuous molecular dynamics simulations, we investigate the molecular organization of King-Webb peptide nanofibers. KW+ and KW- peptides co-assemble into near stoichiometric two-component β-sheet structures as observed by computational simulations and 13C-13C dipolar couplings. A majority of β-strands are aligned with antiparallel nearest neighbors within the β-sheet as previously suggested by Fourier transform infrared spectroscopy measurements. Surprisingly, however, a significant proportion of β-strand neighbors are parallel. While charge-complementary peptides were previously assumed to organize in an ideal (AB)n pattern, dipolar recoupling measurements on isotopically diluted nanofiber samples reveal a non-negligible amount of self-associated (AA and BB) pairs. Furthermore, computational simulations predict these different structures can coexist within the same nanofiber. Our results highlight structural disorder at the molecular level in a charge-complementary peptide system with implications on co-assembling peptide designs.

Peptide coassembly, wherein at least two different peptides interact to form multicomponent nanostructures, is an attractive approach for generating functional biomaterials. Current efforts seek to design pairs of peptides, A and B, that form nanostructures (e.g., β-sheets with ABABA-type β-strand patterning) while resisting self-assembly (e.g., AAAAA-type or BBBBB-type β-sheets). To confer coassembly behavior, most existing designs have been based on highly charged variants of known self-assembling peptides; like-charge repulsion limits self-assembly while opposite-charge attraction promotes coassembly. Recent analyses using solid-state NMR and coarse-grained simulations reveal that preconceived notions of structure and molecular organization are not always correct. This perspective highlights recent advances and key challenges to understanding and controlling peptide coassembly.

Peptides’ hierarchical coassembly into nanostructures enables controllable fabrication of multicomponent biomaterials. In this work, we describe a computational and experimental approach to design pairs of charge-complementary peptides that selectively coassemble into β-sheet nanofibers when mixed together but remain unassembled when isolated separately. The key advance is a peptide coassembly design (PepCAD) algorithm that searches for pairs of coassembling peptides. Six peptide pairs are identified from a pool of ~106 candidates via the PepCAD algorithm and then subjected to DMD/PRIME20 simulations to examine their co-/self-association kinetics. The five pairs that spontaneously aggregate in kinetic simulations selectively coassemble in biophysical experiments, with four forming β-sheet nanofibers and one forming a stable nonfibrillar aggregate. Solid-state NMR, which is applied to characterize the coassembling pairs, suggests that the in silico peptides exhibit a higher degree of structural order than the previously reported CATCH(+/−) peptides.

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.

Myocilin-associated glaucoma is a new addition to the list of diseases linked to protein misfolding and amyloid formation. Single point variants of the ∼257-residue myocilin olfactomedin domain (mOLF) lead to mutant myocilin aggregation. Here, we analyze the 12-residue peptide P1 (GAVVYSGSLYFQ), corresponding to residues 326-337 of mOLF, previously shown to form amyloid fibrils in vitro and in silico. We applied solid-state NMR structural measurements to test the hypothesis that P1 fibrils adopt one of three predicted structures. Our data are consistent with a U-shaped fibril arrangement for P1, one that is related to the U-shape predicted previously in silico. Our data are also consistent with an antiparallel fibril arrangement, likely driven by terminal electrostatics. Our proposed structural model is reminiscent of fibrils formed by the Aβ(1-40) Iowa mutant peptide, but with a different arrangement of molecular turn regions. Taken together, our results strengthen the connection between mOLF fibrils and the broader amylome and contribute to our understanding of the fundamental molecular interactions governing fibril architecture and stability.

Peptide co-assembly is attractive for creating biomaterials with new forms and functions. Emergence of these properties depends on the peptide content of the final assembled structure, which is difficult to predict in multicomponent systems. Here using experiments and simulations we show that charge governs content by affecting propensity for self- and co-association in binary CATCH(+/−) peptide systems. Equimolar mixtures of CATCH(2+/2−), CATCH(4+/4−), and CATCH(6+/6−) formed two-component β-sheets. Solid-state NMR suggested the cationic peptide predominated in the final assemblies. The cationic-to-anionic peptide ratio decreased with increasing charge. CATCH(2+) formed β-sheets when alone, whereas the other peptides remained unassembled. Fibrillization rate increased with peptide charge. The zwitterionic CATCH parent peptide, “Q11”, assembled slowly and only at decreased simulation temperature. These results demonstrate that increasing charge draws complementary peptides together faster, favoring co-assembly, while like-charged molecules repel. We foresee these insights enabling development of co-assembled peptide biomaterials with defined content and predictable properties. Co-assembly of peptide mixtures can yield interesting materials, but predictive understanding of assembly pathways is lacking. Here the self- and co-assembly preferences of oppositely-charged peptides are systematically studied experimentally and by molecular dynamics simulations.

Peptide subunit vaccines increase safety by reducing the risk of off-target responses and improving the specificity of the induced adaptive immune response. The immunogenicity of most soluble peptides, however, is often insufficient to produce robust and lasting immunity. Many biomaterials and delivery vehicles have been developed for peptide antigens to improve immune response while maintaining specificity. Peptide nanoclusters (PNC) are a subunit peptide vaccine material that has shown potential to increase immunogenicity of peptide antigens. PNC are comprised only of crosslinked peptide antigen and have been synthesized from several peptide antigens as small as 8 amino acids in length. However, as with many peptide vaccine biomaterials, synthesis requires adding residues to the peptide and/or engaging amino acids within the antigen epitope covalently to form a stable material. The impact of antigen modifications made to enable biomaterial incorporation or formation is rarely investigated, since the goal of most studies is to compare the soluble antigen with biomaterial form of antigen. This study investigates PNC as a platform vaccine biomaterial to evaluate how peptide modification and biomaterial formation with different crosslinking chemistries affect epitope-specific immune cell presentation and activation. Several types of PNC were synthesized by desolvation from the model peptide epitope SIINFEKL, which is derived from the immunogenic protein ovalbumin. SIINFEKL was altered to include extra residues on each end, strategically chosen to enable multiple conjugation chemistry options for incorporation into PNC. Several crosslinking methods were used to control which functional groups were used to stabilize the PNC, as well as the reducibility of the crosslinking. These variations were evaluated for immune responses and biodistribution following in vivo immunization. All modified antigen formulations still induced comparable immune responses when incorporated into PNC compared to unmodified soluble antigen alone. However, some crosslinking methods led to a significant increase in desirable immune responses while others did not, suggesting that not all PNC were processed the same. These results help guide future peptide vaccine biomaterial design, including PNC and a wide variety of conjugated and self-assembled peptide antigen materials, to maximize and tune the desired immune response.

We present solid-state NMR measurements of β-strand secondary structure and inter-strand organization within a 150-kDa oligomeric aggregate of the 42-residue variant of the Alzheimer's amyloid-β peptide (Aβ(1-42)). We build upon our previous report of a β-strand spanned by residues 30-42, which arranges into an antiparallel β-sheet. New results presented here indicate that there is a second β-strand formed by residues 11-24. Contrary to expectations, NMR data indicate that this second β-strand is organized into a parallel β-sheet despite the co-existence of an antiparallel β-sheet in the same structure. In addition, the in-register parallel β-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 β-sheet. We corroborated this unexpected scheme for β-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.

We present solid-state NMR measurements of β-strand secondary structure and inter-strand organization within a 150 kDa oligomeric aggregate of the 42-residue variant of the Alzheimer’s amyloid-β peptide (Aβ(1-42)). This oligomer is characterized by a structure that cannot be explained by any previously proposed model for aggregated Aβ. We build upon our previous report of a β-strand spanned by residues 30-42, which arranges into an antiparallel β-sheet. New results presented here indicate that there is a second β-strand formed by residues 11-24. We show negative results for NMR experiments designed to reveal antiparallel β-sheets formed by this β-strand. Remarkably, we show that this strand is organized into a parallel β-sheet despite the co-existence of an antiparallel β-sheet in the same structure. In addition, the in-register parallel β-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 3 residues that alternates in direction between adjacent molecules along the β-sheet. We corroborated this unexpected scheme for β-strand organization using multiple 2-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.

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.

Significance Commonly observed secondary structures of proteins, such as α -helices and β -sheets, are built from a trans- amide backbone with residues sampling a single region of the Ramachandran plot. Here we report a secondary structure displayed by biomimetic peptoid polymers in which the backbone exhibits the cis conformation and alternating residues display rotational states of opposed (pseudo)chirality. This structure is linear and untwisted and enables strands to pack densely into extended bilayer nanosheets.

Mutant myocilin aggregation is associated with inherited open angle glaucoma, a prevalent optic neuropathy leading to blindness. Comprehension of mutant myocilin aggregation is of fundamental importance to glaucoma pathogenesis and ties glaucoma to amyloid diseases such as Alzheimer’s. Here we probe the aggregation properties of peptides derived from the myocilin olfactomedin domain. Peptides P1 (residues 326–337) and P3 (residues 426–442) were identified previously to form amyloid. Coarse-grained discontinuous molecular dynamics simulations using the PRIME20 force field (DMD/PRIME20) predict that P1 and P3 are aggregation-prone; P1 consistently forms fibrillar aggregates with parallel in-register β-sheets whereas P3 forms β-sheet-containing aggregates without distinct order. Natural abundance (13)C solid-state NMR spectra validate that aggregated P1 exhibits amyloid signatures and is less heterogeneous than aggregated P3. DMD/PRIME20 simulations provide a viable method to predict peptide aggregation propensities and aggregate’s structure/order which cannot be accessed by bioinformatics or readily attained experimentally.

Peptoid nanosheets are supramolecular protein-mimetic materials that form from amphiphilic polypeptoids with aromatic and ionic side chains. Nanosheets have been studied at the nanometer scale, but the molecular structure has been difficult to probe. We report the use of 13C–13C dipolar recoupling solid-state NMR measurements to reveal the configuration of backbone amide bonds selected by 13C isotopic labeling of adjacent α-carbons. Measurements on the same molecules in the amorphous state and in nanosheets revealed that amide bonds in the center of the amino block of peptoid (NaeNpe)7–(NceNpe)7 (B28) favor the trans configuration in the amorphous state and the cis configuration in the nanosheet. This unexpected result contrasts with previous NMR and theoretical studies of short solvated peptoids. Furthermore, examination of the amide bond at the junction of the two charged blocks within B28 revealed a mixture of both cis and trans configurational states, consistent with the previously predicted brickwork...

Dipeptide derivative molecules can self-assemble into space-filling nanofiber networks at low volume fractions (

An efficient protein-folding pathway leading to target structure, and the avoidance of aggregation, is essential to protein evolution and de novo design; however, design details to achieve efficient folding and avoid aggregation are poorly understood. We report characterization of the thermally-induced aggregate of fibroblast growth factor-1 (FGF-1), a small globular protein, by solid-state NMR. NMR spectra are consistent with residual structure in the aggregate and provide evidence of a structured region that corresponds to the region of the folding nucleus. NMR data on aggregated FGF-1 also indicate the presence of unstructured regions that exhibit hydration-dependent dynamics and suggest that unstructured regions of aggregated FGF-1 lie outside the folding nucleus. Since it is known that regions outside the folding nucleus fold late in the folding pathway, we postulate that these regions unfold early in the unfolding pathway and that the partially folded state is more prone to intermolecular aggregation. This interpretation is further supported by comparison with a designed protein that shares the same FGF-1 folding nucleus sequence, but has different 1° structure outside the folding nucleus, and does not thermally aggregate. The results suggest that design of an efficient folding nucleus, and the avoidance of aggregation in the folding pathway, are potentially separable design criteria - the latter of which could principally focus upon the physicochemical properties of 1° structure outside the folding nucleus.

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 β-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 β-sheet designer peptide assembly.

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 non-specialist. 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 3-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 (13)C and (15)N 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.

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.

Understanding the molecular structures of amyloid-β (Aβ) 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 Aβ(1-42) (the 42-residue variant of the Aβ 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 β-strands. Here we report new solid-state NMR constraints that indicate antiparallel intermolecular alignment of β-strands within the oligomers. Specifically, 150-kDa Aβ(1-42) oligomers with uniform 13C and 15N isotopic labels at I32, M35, G37, and V40 exhibit β-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 Hα and G37 Hα 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 Aβ molecules into oligomers.

Adsorption of the 40-residue Alzheimer’s β-amyloid peptide (Aβ40) on a hydrophobic surface leads to formation of potentially disease-relevant aggregates. Existing techniques are limited in characterizing the adsorbed Aβ40 and producing potentially useful Aβ40 microstructures such as microarrays and microparticles. In this paper, a novel approach based on microcontact printing (μCP) to studying and utilizing adsorption of Aβ40 monomers and fibril fragments on hydrophobic surface of polydimethylsiloxane (PDMS) stamps has been developed. By transferring the adsorbed layer from the stamp to a glass substrate, this approach allows easy measurement of thickness of the adsorbed layer. It also enables characterization of the face of the adsorbed layer in contact with the stamp surface. This face exhibits significant higher thioflavin T fluorescence than the face exposed to water, suggesting β-sheet formation induced by the PDMS surface. The intrinsic stability of the adsorbed layer is evaluated by printing the layer on a water-soluble substrate and exposing it to water vapor or water. Stable particulate microstructures in water are obtained by chemically crosslinking the adsorbed peptides. Moreover, co-micropatterning of the different states of Aβ40 (monomers and fibril fragments) is demonstrated. This μCP-based approach is simple, versatile, and holds potential for various applications.

Solid state NMR measurements on selectively 13C-labeled RADA16-I peptide (COCH3–RADARADARADARADA–NH2) were used to obtain new molecular level information on the conversion of α-helices to β-sheets through self-assembly in the solid state with increasing temperature. Isotopic labeling at the A4 Cβ site enabled rapid detection of 13C NMR signals. Heating to 344–363 K with simultaneous NMR detection allowed production of samples with systematic variation of α-helix and β-strand content. These samples were then probed at room temperature for intermolecular 13C–13C 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 β-strand content infers that β-strands are not observed without association with β-sheets, indicating that β-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 β-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 β-sheets, whereas nanofibers are composed of stacked parallel β-sheets with registry shifts between adjacent β-strands in each β-sheet. These results provide evidence for environment-dependent self-assembly mechanisms for RADA16-I β-sheets as well as new constraints on solid state self-assembled structures, which must be avoided to maximize solution solubility and nanofiber yields. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) are the pathogenic species in Alzheimer's disease (AD). However, detailed structural information on these species remains scarce due to low levels of endogenous Aβ oligomers and uncertainties surrounding current in vitro model systems. Herein, we describe a hydroxyl radical footprinting (HRF) study of Aβ42 fibrils and a stable and homogeneous preparation of Aβ42 oligomers. Specific side chain solvent accessibilities of individual residues in the aggregated and fibril forms of Aβ42 are measured with respect to the same residues of Aβ42 in a fully exposed reference state. These data provide residue specific side chain solvent accessibility protection factors and are used in complement with biophysical characterizations and ss-NMR analyses of these systems. Results are discussed in the context of proposed NMR models of Aβ oligomers and fibrils with implications towards further development of therapeutic and diagnostic strategies.

The properties of femtosecond pulsed laser deposited GaAs nanoclusters were investigated. Nanoclusters of GaAs were produced by laser ablating a single crystal GaAs target in vacuum or Ar gas. Atomic force and transmission electron microscopies showed that most of the clusters were spherical and ranged in diameter from 1 nm to 50 nm, with a peak size distribution between 5 nm and 9 nm, depending on the Ar gas pressure or laser fluence. X-ray diffraction, solid-state nuclear magnetic resonance, Auger electron spectroscopy, electron energy loss spectroscopy, and high-resolution transmission electron microscopy revealed that these nanoclusters were randomly oriented GaAs crystallites. An oxide outer shell of ∼2 nm developed subsequently on the surfaces of the nanocrystals as a result of transportation in air. Unpassivated GaAs nanoclusters exhibited no detectable photoluminescence. After surface passivation, these nanoclusters displayed photoluminescence energies less than that of bulk GaAs from which they were made. Our photoluminescence experiments suggest an abundance of sub-band gap surface states in these GaAs nanocrystals.

Cerebral amyloid angiopathy (CAA), is characterized by the deposition of amyloid beta (Aβ) proteins within the walls of the cerebral vasculature with subsequent aggressive vascular inflammation leading to recurrent hemorrhagic strokes. The objective of the study was to develop theranostic nanovehicles (TNVs) capable of a) targeting cerebovascular amyloid; b) providing magnetic resonance imaging (MRI) contrast for the early detection of CAA; and c) treating cerebrovascular inflammation resulting from CAA. The TNVs comprised of a polymeric nanocore made from Magnevist(®) (MRI contrast agent) conjugated chitosan. The nanocore was also loaded with cyclophosphamide (CYC), an immunosuppressant shown to reduce the cerebrovascular inflammation in CAA. Putrescine modified F(ab’)(2) fragment of anti-amyloid antibody, IgG4.1, (pF(ab’)(2)4.1) was conjugated to the surface of the nanocore to target cerebrovascular amyloid. The average size of the control chitosan nanoparticles (conjugated with albumin and are devoid of Magnevist(®), CYC, and pF(ab’)(2)4.1) was 164 ± 1.2 nm and that of the TNVs was 239 ± 4.1 nm. The zeta potential values of the CCNs and TNVs were 21.6 ± 1.7 mV and 11.9 ± 0.5 mV, respectively. The leakage of Magnevist(®) from the TNVs was a modest 0.2% over 4 days, and the CYC release from the TNVs followed Higuchi’s model that describe sustained drug release from polymeric matrices. The studies conducted in polarized human microvascular endothelial cell monolayers (hCMEC/D3) in vitro as well as in mice in vivo have demonstrated the ability of TNVs to target cerebrovascular amyloid. In addition, the TNVs provided contrast for imaging cerebrovascular amyloid using MRI and single photon emission computed tomography. Moreover, the TNVs were shown to reduce pro-inflammatory cytokine production by the Aβ challenged blood brain barrier (BBB) endothelium more effectively than the cyclophosphamide alone.

The dielectric relaxation behavior of organic liquids was investigated using three representative solvent mixtures between 10 and 70 °C in which the effectiveness of ideal free energy relationships for describing the process of dipolar relaxation was examined. The selected solvent mixtures (nitrobenzene−toluene, ethanol−butanol, and butanol−formamide) incorporate different specific molecular interactions. The dielectric spectra of these solvents and solvent mixtures can be fit to the Debye model to obtain dielectric relaxation times and static dielectric constants as a function of temperature and solution composition. The free energy of activation for the dipolar relaxation process, ΔG, and the Kirkwood correlation factor, g, were determined using these fitting parameters for these solvent systems at various temperatures. The results provide useful descriptions of structures and thermodynamic properties of the solvent mixtures. In particular, ΔG behaves ideally in mixing for nitrobenzene−toluene and ethan...

The designer self-assembling peptide RADA16-I forms nanofiber matrices which have shown great promise for regenerative medicine and three-dimensional cell culture. The RADA16-I amino acid sequence has a β-strand-promoting alternating hydrophobic/charged motif, but arrangement of β-strands into the nanofiber structure has not been previously determined. Here we present a structural model of RADA16-I nanofibers, based on solid-state NMR measurements on samples with different schemes for (13)C isotopic labeling. NMR peak positions and line widths indicate an ordered structure composed of β-strands. The NMR data show that the nanofibers are composed of two stacked β-sheets stabilized by a hydrophobic core formed by alanine side chains, consistent with previous proposals. However, the previously proposed antiparallel β-sheet structure is ruled out by measured (13)C-(13)C dipolar couplings. Instead, neighboring β-strands within β-sheets are parallel, with a registry shift that allows cross-strand staggering of oppositely charged arginine and aspartate side chains. The resulting structural model is compared to nanofiber dimensions observed via images taken by transmission electron microscopy and atomic force microscopy. Multiple NMR peaks for each alanine side chain were observed and could be attributed to multiple configurations of side chain packing within a single scheme for intermolecular packing.

Amyloid-β (Aβ) deposition in the brain vasculature results in cerebral amyloid angiopathy (CAA), which occurs in about 80% of Alzheimer's disease (AD) patients. While Aβ42 predominates parenchymal amyloid plaques in AD brain, Aβ40 is prevalent in the cerebrovascular amyloid. Dutch mutation of Aβ40 (E22Q) promotes aggressive cerebrovascular accumulation and leads to severe CAA in the mutation carriers; knowledge of how DutchAβ40 drives this process more efficiently than Aβ40 could reveal various pathophysiological events that promote CAA. In this study we have demonstrated that DutchAβ40 shows preferential accumulation in the blood-brain-barrier (BBB) endothelial cells due to its inefficient blood-to-brain transcytosis. Consequently, DutchAβ40 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 Aβ40 is not robust enough to exercise such a significant impact on its brain clearance. Hence, the cerebrovascular accumulation of Aβ40 is slow and may require other copathologies to precipitate into CAA. In conclusion, the magnitude of Aβ accumulation in the BBB endothelial cells is a critical factor that promotes CAA; hence, clearing vascular endothelium of Aβ proteins may halt or even reverse CAA.

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 β-hairpins aligned into antiparallel β-sheets. Here, we report evidence from solid-state NMR spectroscopy that supports the presence of the hypothesized β-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.

Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) 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 Aβ(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 Aβ(1-42) oligomers reveal chemical shifts of labeled residues that are indicative of β-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 Aβ oligomers proposed previously by others. For Aβ(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 Aβ(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 β-sheets. Structural similarities and differences between Aβ(1-42) oligomers and fibrils suggest that folded β-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 Aβ(1-42) oligomers and fibrils may explain why this oligomeric state appears off-pathway for monomer self-assembly to fibrils.

Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) are the pathogenic species in Alzheimer's disease (AD). However, detailed structural information on these species remains scarce due to low levels of endogenous Aβ oligomers and uncertainties surrounding current in vitro model systems. Herein, we describe a hydroxyl radical footprinting (HRF) study of Aβ42 monomers, dimers, and specially prepared stable and homogeneous oligomers. Specific side chain solvent accessibilities of individual residues in the folded and aggregated forms of Aβ42 are measured with respect to the same residues of Aβ42 in a fully exposed reference state. These data provide residue specific side chain solvent accessibility protection factors and are used in complement with biophysical characterizations and ss-NMR analyses of the same systems. Results are discussed in the context of proposed NMR models of Aβ oligomers with implications towards further development of therapeutic and diagnostic strategies.

The main objective of the current study was to investigate penetration of cell penetrating peptides (CPPs: TAT, R8, R11, and YKA) through skin intercellular lipids using (31)P magic angle spinning (MAS) solid-state NMR. In vitro skin permeation studies were performed on rat skin, and sections (0-60, 61-120, and 121-180μm) were collected and analyzed for (31)P NMR signal. The concentration-dependent shift of 0, 25, 50, 100, and 200mg/ml of TAT on skin layers, diffusion of TAT, R8, R11, and YKA in the skin and time dependent permeation of R11 was measured on various skin sections using (31)P solid-state NMR. Further, CPPs and CPP-tagged fluorescent dye encapsulate liposomes (FLip) in skin layers were tagged using confocal microscopy. The change in (31)P NMR chemical shift was found to depend monotonically on the amount of CPP applied on skin, with saturation behavior above 100mg/ml CPP concentration. R11 and TAT caused more shift in solid-state NMR peaks compared to other peptides. Furthermore, NMR spectra showed R11 penetration up to 180μm within 30min. The results of the solid-state NMR study were in agreement with confocal microscopy studies. Thus, (31)P solid-state NMR can be used to track CPP penetration into different skin layers.

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 β-sheets through self-assembly in the solid state with increasing temperature. Isotopic labeling at the A4 Cβ 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 β-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 β-strand content infers that β-strands are not observed without association with β-sheets, indicating that β-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 β-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 β-sheets, whereas nanofibers are composed of stacked parallel β-sheets with registry shifts between adjacent β-strands in each β-sheet. These results provide evidence for environment-dependent self-assembly mechanisms for RADA16-I β-sheets as well as new constraints on solid state self-assembled structures, which must be avoided to maximize solution solubility and nanofiber yields.

Azobenzene polymer networks have drawn interests in the area of adaptive materials and structures due to their novel photo-responsive material coupling. These materials undergo a complex light driven molecular conformation change of the azobenzene chromophore when the material is exposed to ultraviolet (UV) or blue-green light. This photoisomerization process is characterized by a molecular conformation change from a rod shaped molecule to a strongly kinked molecule, also known as trans-cis photoisomerization under UV light exposure. Exposure to blue-green light can lead to a trans-cis-trans photoisomerization or a relaxation from the kinked cis state back to the trans rod state. The latter process is of strong interests for adaptive structure applications because the relaxation back to the trans state can be controlled by the orientation of polarized light. When these azobenzene molecules undergo this process in a polymer network, bending and twisting deformation can be controlled by the polarization orientation of the blue-green light. To better understand the distribution of the molecular conformation changes that influences macroscopic polymer deformation, we have conducted solid state Nuclear Magnetic Resonance (ss-NMR) tests on fluorine doped azobenzene polymer networks. Here, we illustrate measurable chemical shifts due to blue light exposure near the 450 nm wavelength using a static ss-NMR probe. The results are compared to ss-NMR at different temperatures using Magic Angle Spinning (MAS) NMR to understand any potential influences of heat relative to photoisomerization.Copyright © 2012 by ASME

We report that synthetic RADA16-I peptide transforms to β-strand secondary structure and develops intermolecular organization into β-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 β-strand secondary structure and assemble into β-sheets. However, temperature dependent FTIR and time dependent WAXD data indicate that bound water may hinder the assembly of β-strands into β-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.

Evidence of size-dependent reconstruction in quantum dots leading to changes in bonding is observed through analysis of the (77)Se{(1)H} cross-polarization magic angle spinning and (77)Se spin-echo solid-state NMR for Cd(77)Se quantum dots. The CP-MAS and spin-echo data indicate discrete surface and core (77)Se sites exist with the QD, in which the surface is comprised of numerous reconstructed lattice planes. Due to the nearly 100% enrichment level for (77)Se, efficient spin coupling is observed between the surface (77)Se and sublayer (77)Se sites due to spin diffusion in the Cd(77)Se quantum dots. The observed chemical shift for the discrete (77)Se sites can be correlated to the effective mass approximation via the Ramsey expression, indicating a 1/r(2) size dependence for the change in chemical shift with size, while a plot of chemical shift versus the inverse band gap is linear. The correlation of NMR shift for the discrete sites allows a valence bond theory interpretation of the size-dependent changes in bonding character within the reconstructed QD. The NMR results provide a structural model for the QDs in which global reconstruction occurs below 4 nm in diameter, while an apparent self-limiting reconstruction process occurs above 4 nm.