Lithium-enriched graphite anode surfaces investigated using nuclear reaction analysis.

Non-destructive Li nuclear reaction analysis techniques were used to profile the Li distribution at the surface of graphitic Li-ion battery anodes. These techniques show that Li concentrations are elevated within 300 nm of the anode surface, even in fully delithiated states. The surface region, which includes the solid electrolyte interphase, contains at least 60% of the total Li irreversibly lost during formation and cycling.

Resonance Energy Transfer in a Genetically Engineered Polypeptide Results in Unanticipated Fluorescence Intensity.

The fluorescence intensity of a C-terminal acceptor chromophore, N-(7-dimethylamino-4-methyl coumarin (DACM), increased proportionally with 280 nm irradiation of an increasing number of donor tryptophan residues located on a ß-sheet forming polypeptide. The fluorescence intensity of the acceptor chromophore increased even as the length of the ß-sheet edge approached 256 Å, well beyond the Förster radius for the tryptophan-acceptor chromophore pair. The folding of the peptides under investigation was verified by circular dichroism (CD) and deep UV resonance Raman experiments. Control experiments showed that the enhancement of DACM fluorescence occurred concomitantly with peptide folding. In other control experiments, the DACM fluorescence intensity of the solutions of tryptophan and DACM did not show any enhancement of DACM fluorescence with increasing tryptophan concentrations. Formation of fibrillar aggregates of the substrate peptides prepared for the fluorescence studies was undetectable by thioflavin T (ThT) fluorescence.

The role of proline-containing peptide triads in ß-sheet formation: A kinetic study.

The design of biomimetic materials through molecular self-assembly is a growing area of modern nanotechnology. With problems of protein folding, self-assembly, and sequence-structure relationships as essential in nanotechnology as in biology, the effect of the nucleation of ß-hairpin formation by proline on the folding process has been investigated in model studies. Previously such studies were limited to investigations of the influence of proline on the formation of turns in short peptide sequences. The effect of proline-based triads on the folding of an 11-kDa amyloidogenic peptide GH6[(GA)3GY(GA)3GE]8 GAH6 (YE8) was investigated by selective substitution of the proline-substituted triads at the γ-turn sites. The folding and fibrillation of the singly proline-substituted polypeptides, e.g., GH6-[(GA)3GY(GA)3GE]7(GA)3GY(GA)3PD-GAH6 (8PD), and doubly proline-substituted polypeptides, e.g., GH6-[(GA)3GY(GA)3GE]3(GA)3GY(GA)3PD[(GA)3GY(GA)3GE]3(GA)3GY(GA)3PD-GAH6 (4,8PD), were directly monitored by circular dichroism and deep UV resonance Raman and fluorescence spectroscopies. These findings were used to identify the essential folding domains, i.e., the minimum number of ß-strands necessary for stable folding. These experimental findings may be especially useful in the design and construction of peptidic materials for a wide range of applications as well as in understanding the mechanisms of folding critical to fibril formation.

Chimera-induced folding: implications for amyloidosis.

The discoveries that non-native proteins have a role in amyloidosis and that multiple protein misfolding diseases can occur concurrently suggest that cross-seeding of amyloidogenic proteins may be central to misfolding. To study this process, a synthetic chimeric amyloidogenic protein (YEHK21-YE8) composed of two components, one that readily folds to form fibrils (YEHK21) and one that does not (YE8), was designed. Secondary structural conformational changes during YEHK21-YE8 aggregation demonstrate that, under the appropriate conditions, YEHK21 is able to induce fibril formation of YE8. The unambiguous demonstration of the induction of folding and fibrillation within a single molecule illuminates the factors controlling this process and hence suggests the importance of those factors in amyloidogenic diseases.

Fibrillation mechanism of a model intrinsically disordered protein revealed by 2D correlation deep UV resonance Raman spectroscopy.

Understanding of numerous biological functions of intrinsically disordered proteins (IDPs) is of significant interest to modern life science research. A large variety of serious debilitating diseases are associated with the malfunction of IDPs including neurodegenerative disorders and systemic amyloidosis. Here we report on the molecular mechanism of amyloid fibrillation of a model IDP (YE8) using 2D correlation deep UV resonance Raman spectroscopy. YE8 is a genetically engineered polypeptide, which is completely unordered at neutral pH yet exhibits all properties of a fibrillogenic protein at low pH. The very first step of the fibrillation process involves structural rearrangements of YE8 at the global structure level without the detectable appearance of secondary structural elements. The formation of ß-sheet species follows the global structural changes and proceeds via the simultaneous formation of turns and ß-strands. The kinetic mechanism revealed is an important new contribution to understanding of the general fibrillation mechanism proposed for IDP.

Metallization of a genetically engineered polypeptide.

Recently, well-ordered biological materials have been exploited to pattern inorganic nanoparticles into linear arrays that are of particular interest for nanoelectronic applications. In this work, a de novo designed E. coli-expressed polypeptide (previously shown to form highly rectilinear, ß-sheet-containing structures) operates as a template for divalent metal cations. EDX and TEM analysis verify the attachment of platinum ions to the histidine-rich fibril surface, which was designed specifically to facilitate attachment of chemical moieties. Following chemical reduction, TEM further confirms the formation of localized zero-valent metal aggregates with sub-nanometer interparticle spacing.

Charge distribution and amyloid fibril formation: insights from genetically engineered model systems.

The influence of electrostatic interactions on protein amyloidogenesis has been investigated using de novo designed repetitive polypeptides YEHK21 [GH6[(GA)3GY(GA)3GE(GA)3GY(GA)3GE]21GAH6] and YE8 [GH6[(GA)3GY(GA)3GE]8GAH6]. The beta-sheet forming polypeptides were designed with identical beta-strands but with variable substitution at the turns that enable precise location of charged residues (Topilina et al. Biopolymers 2007, 86 (4), 261-264; Topilina et al. Biopolymers 2010, submitted for publication; Topilina et al. Biomacromolecules 2006, 7 (4), 1104-11). Solubility, folding, and aggregation of YEHK21 and YE8 were shown to be controlled by charge distribution. Under those conditions favoring the development of charge, YEHK21 and YE8 have significant propensities to form intermolecular beta-sheet assemblies illustrating the potential of charged polypeptide chains to form ordered amyloid aggregates even in the absence of additional environmental factors such as the presence of polyelectrolytes, salts, and so on.

A de novo designed 11 kDa polypeptide: model for amyloidogenic intrinsically disordered proteins.

A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) has a significant number of identical weakly interacting beta-strands with the turns and termini functionalized by charged amino acids to control polypeptide folding and aggregation. YE8 exists in a soluble, disordered form at neutral pH but is responsive to changes in pH and ionic strength. The evolution of YE8 secondary structure has been successfully quantified during all stages of polypeptide fibrillation by deep UV resonance Raman (DUVRR) spectroscopy combined with other morphological, structural, spectral, and tinctorial characterization. The YE8 folding kinetics at pH 3.5 are strongly dependent on polypeptide concentration with a lag phase that can be eliminated by seeding with a solution of folded fibrillar YE8. The lag phase of polypeptide folding is concentration dependent leading to the conclusion that beta-sheet folding of the 11-kDa amyloidogenic polypeptide is completely aggregation driven.

Genetic engineering combined with deep UV resonance Raman spectroscopy for structural characterization of amyloid-like fibrils.

Elucidating the structure of the cross-beta core in large amyloid fibrils is a challenging problem in modern structural biology. For the first time, a set of de novo polypeptides was genetically engineered to form amyloid-like fibrils with similar morphology and yet different strand length. Differential ultraviolet Raman spectroscopy allowed for separation of the spectroscopic signatures of the highly ordered beta-sheet strands and turns of the fibril core. The relationship between Raman frequencies and Ramachandran dihedral angles of the polypeptide backbone indicates the nature of the beta-sheet and turn structural elements.

On the quenching of semiconductor quantum dot photoluminescence by proximal gold nanoparticles.

Luminescent quantum dots (QDs) were proven to be very effective fluorescence resonance energy transfer donors with an array of organic dye acceptors, and several fluorescence resonance energy transfer based biosensing assemblies utilizing QDs have been demonstrated in the past few years. Conversely, gold nanoparticles (Au-NPs) are known for their capacity to induce strong fluorescence quenching of conventional dye donors. Using a rigid variable-length polypeptide as a bifunctional biological linker, we monitor the photoluminescence quenching of CdSe-ZnS QDs by Au-NP acceptors arrayed around the QD surface, where the center-to-center separation distance was varied over a broad range of values (approximately 50-200 Angstrom). We measure the Au-NP-induced quenching rates for such QD conjugates using steady-state and time-resolved fluorescence measurements and examine the results within the context of theoretical treatments based on the Förster dipole-dipole resonance energy transfer, dipole-metal particle energy transfer, and nanosurface energy transfer. Our results indicate that nonradiative quenching of the QD emission by proximal Au-NPs is due to long-distance dipole-metal interactions that extend significantly beyond the classical Förster range, in agreement with previous studies using organic dye-Au-NP donor-acceptor pairs.

Beta-sheet folding of 11-kDa fibrillogenic polypeptide is completely aggregation driven.

A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) was designed and genetically engineered to form antiparallel beta-strands of GAGAGA repeats. Modulation of pH enables control of solubility, folding, and aggregation of YE8 by control of the overall polypeptide charge, a consequence of the protonation or deprotonation of the glutamic acid and histidine residues. YE8 exhibits all the major properties of a fibrillogenic protein providing an excellent model for detailed study of the fibrillation. At neutral pH, YE8 is soluble in disordered form, yet at pH 3.5 folds into a predominantly beta-sheet conformation that is fibrillogenic. Atomic force microscopy and transmission electron microscopy indicated the formation of fibrillar aggregates on well-defined, hydrophobic surfaces. The beta-sheet folding of YE8 exhibited a lag phase that could be eliminated by seeding or stirring. The strong dependence of lag time on polypeptide concentration established the limiting step in aggregation as initiation of beta-sheet folding.

Design and preparation of beta-sheet forming repetitive and block-copolymerized polypeptides.

The design and rapid construction of libraries of genes coding beta-sheet forming repetitive and block-copolymerized polypeptides bearing various C- and N-terminal sequences are described. The design was based on the assembly of DNA cassettes coding for the (GA)3GX amino acid sequence where the (GAGAGA) sequences would constitute the beta-strand units of a larger beta-sheet assembly. The edges of this beta-sheet would be functionalized by the turn-inducing amino acids (GX). The polypeptides were expressed in Escherichia coli using conventional vectors and were purified by Ni-nitriloacetic acid (NTA) chromatography. The correlation of polymer structure with molecular weight was investigated by gel electrophoresis and mass spectrometry. The monomer sequences and post-translational chemical modifications were found to influence the mobility of the polypeptides over the full range of polypeptide molecular weights while the electrophoretic mobility of lower molecular weight polypeptides was more susceptible to C- and N-termini polypeptide modifications.

Reversible thermal denaturation of a 60-kDa genetically engineered beta-sheet polypeptide.

A de novo 687-amino-acid residue polypeptide with a regular 32-amino-acid repeat sequence, (GA)(3)GY(GA)(3)GE(GA)(3)GH(GA)(3)GK, forms large beta-sheet assemblages that exhibit remarkable folding properties and, as well, form fibrillar structures. This construct is an excellent tool to explore the details of beta-sheet formation yielding intimate folding information that is otherwise difficult to obtain and may inform folding studies of naturally occurring materials. The polypeptide assumes a fully folded antiparallel beta-sheet/turn structure at room temperature, and yet is completely and reversibly denatured at 125 degrees C, adopting a predominant polyproline II conformation. Deep ultraviolet Raman spectroscopy indicated that melting/refolding occurred without any spectroscopically distinct intermediates, yet the relaxation kinetics depend on the initial polypeptide state, as would be indicative of a non-two-state process. Thermal denaturation and refolding on cooling appeared to be monoexponential with characteristic times of approximately 1 and approximately 60 min, respectively, indicating no detectable formation of hairpin-type nuclei in the millisecond timescale that could be attributed to nonlocal "nonnative" interactions. The polypeptide folding dynamics agree with a general property of beta-sheet proteins, i.e., initial collapse precedes secondary structure formation. The observed folding is much faster than expected for a protein of this size and could be attributed to a less frustrated free-energy landscape funnel for folding. The polypeptide sequence suggests an important balance between the absence of strong nonnative contacts (salt bridges or hydrophobic collapse) and limited repulsion of charged side chains.

Designer variable repeat length polypeptides as scaffolds for surface immobilization of quantum dots.

We demonstrate the use of a series of engineered, variable-length de novo polypeptides to discretely immobilize luminescent semiconductor nanocrystals or quantum dots (QDs) onto functional surfaces. The polypeptides express N-terminal dicysteine and C-terminal hexahistidine residues that flank a variable number (1, 3, 5, 7, 14, 21, 28, or 35) of core beta-strand repeats, with tyrosine, glutamic acid, histidine, and lysine residues located at the turns. Polypeptides have molecular weights ranging from 4 to 83 kDa and retain a rigid structure based on the antiparallel beta-sheet motif. We first use a series of dye-labeled polypeptides to test and characterize their self-assembly onto hydrophilic CdSe-ZnS QDs using fluorescence resonance energy transfer (FRET). Results indicate that peptides maintain their beta-sheet conformation after self-assembly onto the QD surfaces, regardless of their length. We then immobilize biotinylated derivatives of these polypeptides on a NeutrAvidin-functionalized substrate and use them to capture QDs via specific interactions between the peptides' polyhistidine residues and the nanocrystal surface. We found that each of the polypeptides was able to efficiently capture QDs, with a clear correlation between the density of the surface-tethered peptide and the capacity for nanocrystal capture. The versatility of this capture strategy is highlighted by the creation of a variety of one- and two-dimensional polypeptide-QD structures as well as a self-assembled surface-immobilized FRET-based nutrient sensor.

Bilayer fibril formation by genetically engineered polypeptides: preparation and characterization.

A de novo, genetically engineered 687 residue polypeptide expressed in E. coli has been found to form highly rectilinear, beta-sheet containing fibrillar structures. Tapping-mode atomic force microscopy, deep-UV Raman spectroscopy, and transmission electron microscopy definitively established the tendency of the fibrils to predominantly display an apparently planar bilayer or ribbon assemblage. The ordered self-assembly of designed, extremely repetitive, high molecular weight peptides is a harbinger of the utility of similar materials in nanoscience and engineering applications.

Synthesis of mono- and difluoroacetyltrialkylsilanes and the corresponding enol silyl ethers.

A variety of mono- and difluoroacetylsilanes and the corresponding silyl enol ethers were prepared from trifluoroethanol and chlorotrialkylsilanes in the presence of LDA through retro-Brook rearrangement. Sterically demanding silyl groups, especially those bound to oxygen, resulted in higher yields of difluoroacetylsilanes. The yields of difluoroacetylsilanes were also dramatically affected by the method of the termination of the reaction. Difluorohaloacetylsilanes were prepared from the corresponding difluoroethenyl silyl ethers with electrophilic halogenating reagents in good yields. A gamma-fluorinated beta-diketone 9a was prepared from monofluoroacetyltriisopropylsilane by nucleophilic acylation with methyl trifluoroacetate.

Electrolytic Partial Fluorination of Organic Compounds. 30.(1) Drastic Improvement of Anodic Monofluorination of 2-Substituted 1,3-Oxathiolan-5-ones Using the Novel Fluorine Source Et(4)NF.4HF.

The highly regioselective anodic monofluorination of 2-substituted 1,3-oxathiolan-5-ones was successfully carried out using a novel supporting electrolyte, Et(4)NF.4HF, while use of a conventional supporting electrolyte, Et(3)N.3HF, resulted in no formation or extremely low yields of the fluorinated products.