ABSTRACT
The neurofibrillary tangles of Alzheimer’s disease (AD) are composed mainly of paired helical filaments (PHF) made up of aggregated and hyperphosphorylated tau protein. This axonal microtubule-associated protein normally serves to stabilize microtubules; it is highly soluble in most conditions, but in the disease state it begins to aggregate. The reasons for this unusual behavior are not well understood, but several approaches have been developed to mimic the process in vitro using recombinant tau protein. One problem is how to induce the aggregation of a soluble protein; in the case of tau this can be achieved by polyanionic cofactors. Another problem is how to monitor the aggregation in a time-resolved mode in order to determine the aggregation kinetics. Electron microscopy is useful, because it reveals the nature of the aggregates, but it has a poor time resolution and is usually not quantitative. In the past we have used the fluorescence change of the dye thioflavin S, which occurs upon PHF aggregation, but the caveat is that this is an external additive. In order to find intrinsic signals for monitoring aggregation we have recently turned to modifications of the protein. One particularly useful marker is the amino acid
residue tryptophan (Trp) which can be inserted into the tau sequence ad libitum by site-directed mutagenesis. Tau contains no intrinsic Trp; therefore, the inserted Trp residues provide unique fluorescence signals that report on their immediate vicinity. This property can be exploited to probe the accessibility to solvent, the packing of the protein, the pathway of protein folding and conformational changes.
