Enhanced Photoelectrochemical Water Oxidation on Bismuth Vanadate by Electrodeposition of Amorphous Titanium Dioxide

<i>n</i>-BiVO₄ is a promising semiconductor material for photoelectrochemical water oxidation. Although most thin-film syntheses yield discontinuous BiVO₄ layers, back reduction of photo-oxidized products on the conductive substrate has never been considered as a possible energy loss mechanism in the material. We report that a 15 s electrodeposition of amorphous TiO₂ (<i>a</i>-TiO₂) on W:BiVO₄/F:SnO₂ blocks this undesired back reduction and dramatically improves the photoelectrochemical performance of the electrode. Water oxidation photocurrent increases by up to 5.5 times, and its onset potential shifts negatively by ∼500 mV. In addition to blocking solution-mediated recombination at the substrate, the <i>a</i>-TiO₂ filmwhich is found to lack any photocatalytic activity in itselfis hypothesized to react with surface defects and deactivate them toward surface recombination. The proposed treatment is simple and effective, and it may easily be extended to a wide variety of thin-film photoelectrodes

Ultrafast Time-Resolved Studies on Fluorescein for Recognition Strands Architecture in Amyloid Fibrils

Protein aggregation is associated with numerous devastating diseases such as Alzheimer’s, Parkinson’s, and prion diseases. Development of therapeutics would benefit from knowledge of the structural organization of protein molecules in these amyloid aggregates, particularly in their aqueous biological milieu. However, detailed structural studies to date have been mainly on the solid state and have required large quantities of purified aggregate. Moreover, these conventional methods require the aggregated assembly to remain structurally stable over days or weeks required to perform the experiment, whereas the pathologically relevant species of in vivo aggregates may be shorter lived. Here, we show the organization of protein chains in dissolved amyloid aggregates can be readily determined spectroscopically using minute quantities of fluorescein-labeled protein segments in a matter of minutes. Specifically, we investigated the possibility of using the ultrafast dynamics of fluorescein to distinguish among three categories of β-sheet geometry: (<b>1</b>) antiparallel in-register, (<b>2</b>) parallel in-register, or (<b>3</b>) antiparallel out-of-register. Fluorescein, the most commonly used staining dye in biology and medicine, was covalently attached to the N-termini of peptide sequences selected from a library of known amyloid crystal structures. We investigated the aggregates in solution using steady-state and time-resolved absorption and fluorescence spectroscopy. We found that the dynamics of fluorescein relaxation from the excited state revealed amyloid structure-specific information. Particularly, the nonfluorescent cation form of fluorescein showed remarkable sensitivity to local environments created during aggregation. We demonstrate that time-resolved absorption is capable of differentiating strand organization in β-sheet aggregates when strong intermolecular coupling between chromophores occurs. This approach can be useful in optical recognition of specific fibril architectures of amyloid aggregates

X-ray Crystallographic Structure of an Artificial β-Sheet Dimer

This paper describes the X-ray crystallographic structure of a designed cyclic β-sheet peptide that forms a well-defined hydrogen-bonded dimer that mimics β-sheet dimers formed by proteins. The 54-membered ring macrocyclic peptide (<b>1a</b>) contains molecular template and turn units that induce β-sheet structure in a heptapeptide strand that forms the dimerization interface. The X-ray crystallographic structure reveals the structures of the two “Hao” amino acids that help template the β-sheet structure and the two δ-linked ornithine turn units that link the Hao-containing template to the heptapeptide β-strand. The Hao amino acids adopt a conformation that resembles a tripeptide in a β-strand conformation, with one edge of the Hao unit presenting an alternating array of hydrogen-bond donor and acceptor groups in the same pattern as that of a tripeptide β-strand. The δ-linked ornithines adopt a conformation that resembles a hydrogen-bonded β-turn, in which the ornithine takes the place of the <i>i</i>+1 and <i>i</i>+2 residues. The dimers formed by macrocyclic β-sheet <b>1a</b> resemble the dimers of many proteins, such as defensin HNP-3, the λ-Cro repressor, interleukin 8, and the ribonuclease H domain of HIV-1 reverse transcriptase. The dimers of <b>1a</b> self-assemble in the solid state into a barrel-shaped trimer of dimers in which the three dimers are arranged in a triangular fashion. Molecular modeling in which one of the three dimers is removed and the remaining two dimers are aligned face-to-face provides a model of the dimers of dimers of closely related macrocyclic β-sheet peptides that were observed in solution

Macrocyclic β-Sheet Peptides That Inhibit the Aggregation of a Tau-Protein-Derived Hexapeptide

This paper describes studies of a series of macrocyclic β-sheet peptides <b>1</b> that inhibit the aggregation of a tau-protein-derived peptide. The macrocyclic β-sheet peptides comprise a pentapeptide “upper” strand, two δ-linked ornithine turn units, and a “lower” strand comprising two additional residues and the β-sheet peptidomimetic template “Hao”. The tau-derived peptide Ac-VQIVYK-NH₂ (AcPHF6) aggregates in solution through β-sheet interactions to form straight and twisted filaments similar to those formed by tau protein in Alzheimer’s neurofibrillary tangles. Macrocycles <b>1</b> containing the pentapeptide VQIVY in the “upper” strand delay and suppress the onset of aggregation of the AcPHF6 peptide. Inhibition is particularly pronounced in macrocycles <b>1a</b>, <b>1d</b>, and <b>1f</b>, in which the two residues in the “lower” strand provide a pattern of hydrophobicity and hydrophilicity that matches that of the pentapeptide “upper” strand. Inhibition varies strongly with the concentration of these macrocycles, suggesting that it is cooperative. Macrocycle <b>1b</b> containing the pentapeptide QIVYK shows little inhibition, suggesting the possibility of a preferred direction of growth of AcPHF6 β-sheets. On the basis of these studies, a model is proposed in which the AcPHF6 amyloid grows as a layered pair of β-sheets and in which growth is blocked by a pair of macrocycles that cap the growing paired hydrogen-bonding edges. This model provides a provocative and appealing target for future inhibitor design

Amyloid β‑Protein C‑Terminal Fragments: Formation of Cylindrins and β‑Barrels

In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer’s disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24–34), Aβ(25–35) and Aβ(26–36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins

Heterologous Expression of Mycobacterial Esx Complexes in <i>Escherichia coli</i> for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions

<div><p>The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in <i>Escherichia coli</i>, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the <i>Mycobacterium abscessus</i> EsxEF, <i>M</i>. <i>smegmatis</i> EsxGH, and <i>M. tuberculosis</i> EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.</p></div

Top five highest non-redundant results for the DALI search using the two chains of the Esx complexes as a single chain search model.

a<p>If known.</p>b<p>The number in parentheses is the total length of the similar structure.</p

Data collection statistics for <i>M</i>. <i>abscessus</i> EsxEF, <i>M</i>. <i>smegmatis</i> EsxGH, and <i>M</i>. <i>tuberculosis</i> EsxOP complexes.

*<p>Numbers in parentheses refer to the high resolution data shell.</p

Schematic of Esx complex coexpression strategies.

<p>Bicistronic operons encoding the Esx complex genes, separated by a naturally occurring intergenic region of variable length, were cloned into three different expression vectors. (A) The two subunits are coexpressed from a single bicistronic transcript with an N-terminal His₆ tag and TEV protease site on the CFP-10 homolog. (B) The two subunits are coexpressed from a single bicistronic transcipt with an N-terminal MBP fusion with His₆ tag and TEV protease site on the CFP-10 homolog. (C) The two subunits are coexpressed from a single bicistronic transcript with an N-terminal MBP fusion with TEV protease site on the CFP-10 homolog and a C-terminal His₆ tag on the ESAT-6 homolog. Concurrent expression of TEV protease (TEVp) cleaves the MBP moiety from the CFP-10 homolog intracellularly at the TEV protease site positioned between the MBP C-terminus and CFP-10 N-terminus.</p

Structural homologs of the mycobacterial Esx complexes described in this study. Non-redundant targets in the first 200 results of the DALI search results are listed.

a<p>In addition to protein name whether the structurally similar chain is of a CFP-10 or ESAT-6 homolog is noted. Alternatively, if the similar structure is of a homodimeric Esx complex or of a PE protein this information is provided.</p>b<p>The number in parentheses is the total length of the similar structure.</p