Labile Zinc-Assisted Biological Phosphate Chemosensing and Related Molecular Logic Gating Interpretations

Herein, molecular fluorescence ‘OFF–ON’ behavior with aqueous addition of biological phosphate and Zn²⁺ is studied with Zn₂(<i>slys</i>)₂Cl₂ [H₂<i>slys</i> = 6-amino-2-{(2-hydroxybenzylidene)­amino}­hexanoic acid], a fluorescent water-soluble complex, using various spectroscopic tools (e.g., ³¹P NMR, UV–vis, emission, and CD spectroscopy) at the micromolar level. Adduct-dependent fluorescence intensity changes can be interpreted as a two-input (cation/anion) implication molecular logic gating system. A displacement study of PPi from the dizinc complex is also reported. Diphosphate and triphosphate addition/displacements were also studied. ³¹P NMR spectroscopy shows gradual NMR peak shifts from bound ADP/GDP to free ADP/GDP with increasing [PPi]. In the emission spectrum, fluorescence quenching is shown: CD signal maxima decrease with addition of PPi. These displacement events are also tested with triphosphates (ATP, GTP), and their binding strength/displacement ability over ADP/GDP is quantified: PPi > ATP ≈ GTP (3.35 ± 0.77 × 10⁴ M^–1 for PPi, 7.73 ± 1.79 × 10³ M^–1 for ATP, 9.21 ± 2.88 × 10³ M^–1 for GTP over <b>1</b>·ADP). Many anions and cations were also screened for selectivity. Tubulin polymerization was assayed in the presence of <b>1</b> and its copper analogue which reflected a slight inhibition in polymerization

Facile &quot;stop codon&quot; method reveals elevated neuronal toxicity by discrete S87p-alpha-synuclein oligomers

Herein, a new method for preparing phosphorylated proteins at specific sites has been applied to alpha-synuclein (alpha-Syn). Three different alpha-Syn species phosphorylated at Serine 87 (S87p-alpha-Syn), Serine 129 (S129p-alpha-Syn) and Serine 87/129 (S87p,129p-alpha-Syn) were prepared through the &apos;stop codon&apos; method and verified by LC/MS/MS and immunoblotting. Each type of phosphorylated alpha-Syn was tested for oligomerization trends and cellular toxicity with dopamine (DA), Cu2+ ions and pyridoxal 5&apos;-phosphate. Aggregation trends induced by DA or DA/Cu2+ were similar between phosphorylated and non-phosphorylated alpha-Syn in SDS-PAGE. However, except for the monomer, phosphorylated oligomers showed higher toxicity than the non-phosphorylated alpha-Syn (Np-alpha-Syn) oligomers via WST-1 assays when tested on SH-SY5Y human neuroblastoma cells. In particular, S87p-alpha-Syn and S87p,129p-alpha-Syn oligomers induced by DA/Cu2+, showed higher toxicity than did S129p-alpha-Syn. When alpha-Syn was treated with pyridoxal 5&apos;-phosphate in the presence of DA or Cu2+ to determine aggregation effects, high inhibition effects were shown in both non-phosphorylated and phosphorylated versions. alpha-Syn co-incubated with DA or DA/Cu2+ showed less cellular toxicity upon pyridoxal 5&apos;-phosphate treatment, especially in the case of DA-induced Np-alpha-Syn. This study supports that phosphorylated oligomers of alpha-Syn at residue 87 can contribute to neuronal toxicity and the pyridoxal 5&apos;-phosphate can be used as an inhibitor for alpha-Syn aggregation. (C) 2013 Elsevier Inc. All rights reserved.N