Conformational basis for asymmetric seeding barrier in filaments of three- and four-repeat tau

Tau pathology in Alzheimer's disease is intimately linked to the deposition of proteinacious filaments, which akin to infectious prions, have been proposed to spread via seeded conversion. Here we use double electron-electron resonance (DEER) spectroscopy in combination with extensive computational analysis to show that filaments of three- (3R) and four-repeat (4R) tau are conformationally distinct. Distance measurements between spin labels in the third repeat, reveal tau amyloid filaments as ensembles of known β-strand-turn-β-strand U-turn motifs. Whereas filaments seeded with 3R tau are structurally homogeneous, filaments seeded with 4R tau are heterogeneous, composed of at least three distinct conformers. These findings establish a molecular basis for the seeding barrier between different tau isoforms and offer a new powerful approach for investigating the composition and dynamics of amyloid fibril ensembles.     

Click strategies for single-molecule protein fluorescence

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.     

Second-Generation Covalent TMP-Tag for Live Cell Imaging

Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology.     

Mechanistic insight on the catecholase activity of dinuclear copper complexes with distant metal centers

The catecholase activity of two dinuclear Cu(II) complexes with distant metal centers is discussed together with solid state and solution studies. The crystal structure for one of them, [Cu(2)(diep)(H(2)O)(4)](ClO(4))(4)·2H(2)O, is described, showing the two copper ions are 7.457 ? apart and in a square pyramidal coordination. Both complexes display a weak antiferromagnetic coupling in the solid state that is manifest in the dimer EPR spectra obtained in frozen solution. The pH-potentiometric speciation performed in 1:1 MeOH-H(2)O allowed the assignment of hydrolyzed copper species as those catalytically active in the oxidation of 3,5-di-tert-butylcatechol (DTBC). The kinetic measurements led us to propose behavior consistent with Michaelis-Menten plus a linear dependence of the initial rate on [DTBC]. This can be associated with the presence of more than one catalytically active species, which is consistent with the evidence of several differently hydrolyzed species shown in the predominance diagrams. Product characterization studies led to establishing the formation of hydrogen peroxide during the catalytic cycle, while semiquinone and superoxide radicals were detected by EPR spectroscopy, supporting one-electron transference at each of the copper centers.     

On coderivatives and Lipschitzian properties of the dual pair in optimization

In this paper, we apply the concept of coderivative and other tools from the generalized differentiation theory for set-valued mappings to study the stability of the feasible sets of both the primal and the dual problem in infinite-dimensional linear optimization with infinitely many explicit constraints and an additional conic constraint. After providing some specific duality results for our dual pair, we study the Lipschitz-like property of both mappings and also give bounds for the associated Lipschitz moduli. The situation for the dual shows much more involved than the case of the primal problem.     

Derivation of the near-surface dielectric function of amorphous silicon from photoelectron loss spectra

The near-surface dielectric function ε(?ω) of hydrogenated amorphous silicon (a-Si:H) films has been derived from X-ray photoelectron energy-loss spectra, over the energy range 0–40 eV. Removal of low lying single-electron excitations is a prerequisite step to proceed to the derivation of the single plasmon energy loss function Im[? 1/ε(?ω)] due to collective electron oscillations. Several methods are compared to separate interband transitions from bulk or surface plasmons excitation. The shape of interband excitation loss in the range 1–10 eV can be described by a Henke function; alternatively, its removal using a sigmoid weighting function is a low-noise and reliable method. After deconvolution of multiple plasmon losses and self-consistent elimination of surface plasmon excitation, the single plasmon loss distribution allows recovery of optical (ellipsometry) data measured in the near-UV to visible range.     

A new phenanthrene-based bis-oxime chemosensor for Fe(III) and Cr(III) discrimination

The synthesis of two new 2,7-disubstituted phenanthrene-based bis oximes is described. The ability of these two compound for complexing heavy metal cations have been studied and complexation constants and complex stoichiometry for Cr³⁺ and Fe³⁺ complex have been determined. The fluorescent properties of ligand 2 make this compound able to act as a sensor able to discriminate between Cr³⁺ and Fe³⁺. Detection limits for these two cations have been evaluated.     

Development of a headspace-solid phase microextraction-gas chromatography method to determine organohalogen contamination in drinking water

The formation of organohalogen compounds in waters treated by chlorination has drawn increasing scientific attention due to the potentially hazardous health effects of this class of substances. Today, chlorination is the most widely used technology for civil water disinfection. In this study, headspace-solid phase microextraction coupled with GC-electron capture detector was used to determine organohalogen compounds in drinking water sampled from aqueducts and artesian wells in Italy. Experimental parameters, such as sample volume, stirring, salting out, extraction temperature, and extraction time, were evaluated and optimized. The LODs ranged from 1 to 10 ng/L and LOQs from 5 to 50 ng/L. A linear response was confirmed by correlation coefficients ranging from 0.9443 to 0.9999. Quantifiable organohalogen residues were found in 11 water samples, with concentration up to 11.3 +/- 0.5 microg/L for the sum of all trihalomethanes and 0.66 +/- 0.03 microg/L for the sum of trichloroethylene and tetrachloroethylene. These concentrations are lower than the current regulatory limits in Italy.