Site-specific C-terminal fluorescent labeling of Tau protein

Formation of Tau protein aggregates in neurons is a pathological hallmark of several neurodegenerative diseases, including Alzheimers disease. Fluorescently-labeled Tau protein is therefore useful to study the aggregation of these pathological proteins and to identify potential therapeutic targets. Conventionally, cysteine residues are used for labeling Tau proteins, however, the full-length Tau isoform contains two cysteine residues in the microtubule-binding region, which are implicated in Tau aggregation by forming intermolecular disulfide bonds. In order to prevent the fluorescent label from disturbing the microtubule binding region, we developed a strategy to fluorescently label Tau at its C-terminus while leaving cysteine residues unperturbed. We took advantage of a Sortase A-mediated transpeptidation approach to bind a short peptide (GGGH6-Alexa647) with a His-tag and a covalently attached Alexa 647 fluorophore to the C-terminus of Tau. This reaction relies on the presence of a Sortase recognition motif (LPXTG), which we attached to the C-terminus of recombinantly expressed Tau. We determined the possible effects of the resulting C-terminal modification on the secondary structure of Tau protein, its aggregation kinetics, and fibril morphology compared to the unlabeled native Tau protein in vitro. The results showed no significant differences between the native and C-terminally labeled Tau monomer with regard to aggregation kinetics, secondary structure, and morphology.