Anant K. Paravastu

Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils

We describe a full structural model for amyloid fibrils formed by the 40-residue β-amyloid peptide associated with Alzheimers disease (Aβ1–40), based on numerous constraints from solid state NMR and electron microscopy. This model applies specifically to fibrils with a periodically twisted morphology, with twist period equal to 120 ± 20 nm (defined as the distance between apparent minima in fibril width in negatively stained transmission electron microscope images). The structure has threefold symmetry about the fibril growth axis, implied by mass-per-length data and the observation of a single set of 13C NMR signals. Comparison with a previously reported model for Aβ1–40 fibrils with a qualitatively different, striated ribbon morphology reveals the molecular basis for polymorphism. At the molecular level, the 2 Aβ1–40 fibril morphologies differ in overall symmetry (twofold vs. threefold), the conformation of non-β-strand segments, and certain quaternary contacts. Both morphologies contain in-register parallel β-sheets, constructed from nearly the same β-strand segments. Because twisted and striated ribbon morphologies are also observed for amyloid fibrils formed by other polypeptides, such as the amylin peptide associated with type 2 diabetes, these structural variations may have general implications.

Seeded growth of β-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 β-amyloid (Aβ1–40) peptides have shown that the molecular structure of Aβ1–40 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 β-amyloid fibrils that develop in Alzheimers 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 Aβ1–40 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 Aβ1–40 fibrils most likely reflect structures that are present in AD brain. Solid-state 13C 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. 13C chemical shifts and other NMR data indicate that the predominant molecular structure in brain-seeded fibrils differs from the structures of purely synthetic Aβ1–40 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.

The Alzheimer's Amyloid-β(1–42) Peptide Forms Off-Pathway Oligomers and Fibrils That Are Distinguished Structurally by Intermolecular Organization

Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) initiate the neurotoxicity that eventually leads to dementia in Alzheimers 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.

Molecular Structure of RADA16-I Designer Self-Assembling Peptide Nanofibers

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.

Frequency-selective homonuclear dipolar recoupling in solid state NMR

We introduce a new approach to frequency-selective homonuclear dipolar recoupling in solid state nuclear magnetic resonance (NMR) with magic-angle spinning (MAS). This approach, to which we give the acronym SEASHORE, employs alternating periods of double-quantum recoupling and chemical shift evolution to produce phase modulations of the recoupled dipole-dipole interactions that average out undesired couplings, leaving only dipole-dipole couplings between nuclear spins with a selected pair of NMR frequencies. In principle, SEASHORE is applicable to systems with arbitrary coupling strengths and arbitrary sets of NMR frequencies. Arbitrary MAS frequencies are also possible, subject only to restrictions imposed by the pulse sequence chosen for double-quantum recoupling. We demonstrate the efficacy of SEASHORE in experimental (13)C NMR measurements of frequency-selective polarization transfer in uniformly (15)N, (13)C-labeled L-valine powder and frequency-selective intermolecular polarization transfer in amyloid fibrils formed by a synthetic decapeptide containing uniformly (15)N, (13)C-labeled residues.

Size- and Site-Dependent Reconstruction in CdSe QDs Evidenced by 77Se{1H} CP-MAS NMR Spectroscopy

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.

Probing the molecular structure of antimicrobial peptide-mediated silica condensation using X-ray photoelectron spectroscopy

The antimicrobial peptide KSL (KKVVFKVKFK) mediates the rapid condensation of tetramethyl orthosilicate to form silica nanoparticles. X-ray photoelectron spectroscopy (XPS) was employed to identify the molecular interactions between protein and silica on the surface of nanoparticles containing antimicrobial peptide and silica. Comparative high resolution spectral analysis between KSL peptide and KSL-catalyzed silica nanoparticles revealed that imidates are present in the KSL peptide backbone after silica formation. Supporting evidence for the presence of an imidate is provided by FTIR spectroscopic analysis of the amide I and V bands. XPS analysis also shows that side-chain amines of lysine do not interact with the silica product and the lack of association is supported further by 15N-29Si REDOR NMR. Quantitative analysis of XPS elemental spectra determined the silica O:Si ratio is 3.6 : 1, suggesting that the nuclei (sol) particles are not highly condensed structures. Results were supported using 29Si CPMAS NMR to show that the majority of the silica is Q2 groups. A proposed mechanism of rapid silicification with involvement of a peptide imidate is presented.

Physical characterization of functionalized spider silk: electronic and sensing properties

Abstract This work explores functional, fundamental and applied aspects of naturally harvested spider silk fibers. Natural silk is a protein polymer where different amino acids control the physical properties of fibroin bundles, producing, for example, combinations of β-sheet (crystalline) and amorphous (helical) structural regions. This complexity presents opportunities for functional modification to obtain new types of material properties. Electrical conductivity is the starting point of this investigation, where the insulating nature of neat silk under ambient conditions is described first. Modification of the conductivity by humidity, exposure to polar solvents, iodine doping, pyrolization and deposition of a thin metallic film are explored next. The conductivity increases exponentially with relative humidity and/or solvent, whereas only an incremental increase occurs after iodine doping. In contrast, iodine doping, optimal at 70 °C, has a strong effect on the morphology of silk bundles (increasing their size), on the process of pyrolization (suppressing mass loss rates) and on the resulting carbonized fiber structure (that becomes more robust against bending and strain). The effects of iodine doping and other functional parameters (vacuum and thin film coating) motivated an investigation with magic angle spinning nuclear magnetic resonance (MAS-NMR) to monitor doping-induced changes in the amino acid-protein backbone signature. MAS-NMR revealed a moderate effect of iodine on the helical and β-sheet structures, and a lesser effect of gold sputtering. The effects of iodine doping were further probed by Fourier transform infrared (FTIR) spectroscopy, revealing a partial transformation of β-sheet-to-amorphous constituency. A model is proposed, based on the findings from the MAS-NMR and FTIR, which involves iodine-induced changes in the silk fibroin bundle environment that can account for the altered physical properties. Finally, proof-of-concept applications of functionalized spider silk are presented for thermoelectric (Seebeck) effects and incandescence in iodine-doped pyrolized silk fibers, and metallic conductivity and flexibility of micron-sized gold-sputtered silk fibers. In the latter case, we demonstrate the application of gold-sputtered neat spider silk to make four-terminal, flexible, ohmic contacts to organic superconductor samples.

Properties of GaAs nanoclusters deposited by a femtosecond laser

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.

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

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.