title: Electrochemical investigations into kinase-catalyzed transformations of tau protein.
abstract: 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.
authors.affiliation: Department of Physical and Environmental Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, ON, M1C1A4, Canada.
authors.forename: Meghan K
authors.initials: MK
authors.lastname: Rains
authors.forename: Sanela
authors.initials: S
authors.lastname: Martić
authors.forename: Daniel
authors.initials: D
authors.lastname: Freeman
authors.forename: Heinz Bernhard
authors.initials: HB
authors.lastname: Kraatz
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D055664:Electrochemical Techniques / Q000379:methods; D005296:Ferrous Compounds / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D000075163:Metallocenes; D010766:Phosphorylation; D056951:Photoelectron Spectroscopy / Q000379:methods; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D013499:Surface Properties; D016875:tau Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005296:Ferrous Compounds; C000715470:MAS1 protein, human; D000075163:Metallocenes; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D016875:tau Proteins; D006046:Gold; D000255:Adenosine Triphosphate; C095903:tau-tubulin kinase; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D038362:Glycogen Synthase Kinase 3; C004998:ferrocene