Electrochemical Investigations into Kinase-Catalyzed Transformations of Tau Protein

The formation of neurofibrillary tangles by hyperphosphorylated tau is a well-recognized hallmark of Alzheimer’s disease. Resulting from malfunctioning protein kinases, hyperphosphorylated tau is unable to bind microtubules properly, causing it to self-associate and aggregate. The effects of tau phosphorylation on tau conformation and aggregation are still largely unexplored. The conformational analysis of tau and its hyperphosphorylated forms is usually performed by a variety of spectroscopic techniques, all of which require ample sample concentrations and/or volumes. Here we report on the use of surface based electrochemical techniques that allow for detection of conformational changes and orientation of tau protein as a function of tau phosphorylation by tyrosine and serine/threonine kinases. The electrochemical methods utilize 5′-γ-ferrocenyl adenosine triphosphate (Fc-ATP) derivative as a cosubstrate and tau immobilized on gold surface to probe the role of the following protein kinases: Sarcoma related kinase (Src), Abelson tyrosine kinase (Abl), tau-tubulin kinase (TTBK), proto-oncogene tyrosine protein kinase Fyn (Fyn), and glycogen synthase kinase 3-β (Gsk-3β). The single kinase and sequential kinase-catalyzed Fc-phosphorylations modulate the electrochemical signal, pointing to the dramatic changes around the Fc group in the Fc-phosphorylated tau films. The location and orientation of the Fc-group in Fc-tau film was investigated by the surface plasmon resonance based on antiferrocene antibodies. Additional surface characterization of the Fc-tau films by time-of-flight secondary ion-mass spectrometry and X-ray photoelectron spectroscopy revealed that Fc-phosphorylations influence the tau orientation and conformation on surfaces. When Fc-phosphorylations were performed in solution, the subsequently immobilized Fc-tau exhibited similar trends. This study illustrates the validity and the utility of the labeled electrochemical approach for probing the changes in protein film properties, conformation, and orientation as a function of the enzymatically catalyzed modifications

Versatile Strategy for Biochemical, Electrochemical and Immunoarray Detection of Protein Phosphorylations

Protein kinases catalyze the phosphorylation of cellular proteins involved in the regulation of many cellular processes and have emerged as promising targets for the treatment of several diseases. Conventional assays to monitor protein kinase activity are limited because they typically rely on transfer of radioactive phosphate or phospho-specific antibodies that recognize specific substrates or sequence motifs. To overcome the limitations of conventional assays, we have developed a versatile approach based on transfer of ferrocene-phosphate that can be readily monitored using electrochemical detection or detection with antiferrocene antibodies in an immunoarray format. This assay is readily adapted to multiplex arrays and can be employed for monitoring kinase activity in complex mixtures and for kinase inhibitor profiling

Synthesis and Surface Investigations of N-Substituted 2,5-Dithio-7-azabicyclo[2.2.1]heptanes on Gold Surfaces

The reaction of various primary amines and 2,5-dihydroxy-1,4-dithiane in the presence of a catalytic amount of Mg­(II) in distilled water provided a series of N-substituted 2,5-dithia-7-azabicyclo[2.2.1]­heptanes. The adsorption profiles of the sulfur-containing heterocycles on gold surfaces have been explored by time-of-flight secondary ion mass spectrometry (TOF-SIMS), X-ray photoelectron spectroscopy (XPS), and electrochemistry. SIMS data indicated that these novel bicyclic sulfides interact with gold surfaces favorably, independent of the N-substitution, with minimal fragmentation. An XPS study revealed the three component core levels of S 2p with binding energies at 161, 162, and 163 eV, indicating a combination of the bound and unbound sulfur species. Using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), we found the efficient adsorption of heterocycles onto gold and the formation of densely packed films for alkyl and phenyl analogues. However, the adsorption and film packing properties were greatly compromised by an N<i>-</i>pyridyl substitution. The findings indicate that the surface behavior of N-substituted 2,5-dithia-7-azabicyclo[2.2.1]­heptanes varies with respect to the N-substitution and the nature of the substituent, suggesting that the adsorption profiles and the film packing of bicyclic sulfides on gold surfaces are highly dependent on the binding interface and the molecular orientation