Much of the insights into tau conformation and aggregation have been obtained from in vitro experiments on recombinant or brain-derived proteins using biochemical and biophysical tools. Those used in this thesis are introduced here. Kinetic assays – using fluorescent dyes
Tau aggregation is rate-dependent: there are different reaction phases that can be monitored kinetically. As tau on its own does not give measurable chemical signals during aggregation (Sahara et al., 2007; Sui et al., 2015), fluorescence changes in the presence of specific chemicals that bind tau are used to estimate the protein’s aggregation. Probably the most widely used is the benzothiazole salt thioflavin T (ThT) or its alternative thioflavine S (ThS), which is a mixture of several sulphonated compounds. When bound to tau monomers, ThT-tau and ThS-tau interactions lead to spectroscopic changes that alter the reported fluorescence signal. Following a brief lag phase, an exponential increase in fluorescence is recorded which is believed to indicate cumulative aggregation until filament formation when the curve reaches a plateau (Biancalana and Koide,
2010). Nonetheless, this property is dependent on the forms of tau used, buffer conditions, spectrophotometer and the tau-ThT/S ratio (Xue et al., 2017). The principle of ThT/S binding to tau filaments has been applied in clinical histopathology to diagnose tauopathies (Bussière et al., 2004; Rajamohamedsait and Sigurdsson, 2012). The mechanism of ThT/S binding to tau is not fully understood: one model suggests that ThT binds to amino acid side surface chains that are arranged in parallel to the b-sheet axis of tau filaments (Biancalana and Koide, 2010). Binding to proteinopathic aggregates can drastically increase the fluorescence intensities of both ThT and ThS, but the main difference is that a corresponding forward shift in emission spectrum is observed for ThT but not ThS for which no change in the excitation or the emission spectra is recorded (Groenning, 2010; LeVine, 1999). This results in a consistently high background fluorescence in the case of ThS, making it unsuitable for quantitative analysis (LeVine, 1999).
Secondary structure determination: circular dichroism (CD)
CD spectroscopy is routinely used to ascertain the structural transition of tau from its random coiled conformation to b-sheet formation. A CD spectrum is obtained due to a sample’s differential absorption of left and right circularly polarised light. The extent to which a test molecule absorbs the two light waves which are arranged 90 O out of phase leads to the generation of an electrical field signal. The resultant differences in the electric field generated by the clockwise- and anti- clockwise-facing lights is calculated to give the CD readout at each wavelength. Signature CD spectra measured in the far UV range differ for given proteins depending on their predominant secondary structure content (Fig. 1.8). Unfolded proteins, such as monomeric tau, have negative peaks at 198 – 200 nm, b-sheet- enriched proteins like PHFs have negative peaks at ~220 nm, whilst soluble aggregated tau (e.g., oligomers) have an intermediate negative peak between 200 and 220 nm. Other proteins with majority a-helix content have two negative peaks at ~210 nm and ~220 nm (Whitmore and Wallace, 2008). CD-resolved secondary structure content can be analysed by comparing spectral signals to those in given databases (Greenfield, 2006; Kelly et al., 2005).
Ultrastructure of aggregated species: atomic force microscopy (AFM) and transmission electron microscopy (TEM)
AFM and TEM are similar techniques used to probe the ultrastructural properties of aggregated tau. AFM involves three-dimensional scanning of a sample surface with a cantilever-suspended flexible probe. Deflection of the cantilever occurs in response to probe-sample surface contacts on a piezoelectric scanner, leading to parallel images formed along the probe tracks. These images are then projected by laser signals onto photodiode detectors and processed with specific algorithms. There are three main AFM modes, depending on probe-sample interactions: contact, non-contact and tapping. Tapping mode imaging is often preferred as it eliminates the disadvantages of the other methods regarding poor resolution and frictional forces that can damage sample surfaces (Carvalho and Santos, 2012; Dufrêne, 2002).
In TEM, an electron beam passes through the sample and the micrometer-scale image formed based on electron-sample interactions is magnified to enhance resolution. As complementary techniques, TEM and AFM compensate for their shortcomings. For example, heavy metal staining in TEM can disrupt some nanoscale details. This challenge can be addressed by using AFM which does not use sample staining and therefore retains samples in their native state. Moreover, small aggregated proteins (e.g. oligomers made of <10-20 monomers) whose imaging can be problematic with TEM can be easily done with AFM. Nonetheless, AFM imaging is extremely slow and gives poor image resolutions. On the contrary, TEM images have better resolution and are achievable within shorter times (Tinker-Mill et al., 2014).
Dynamic light scattering (DLS)
DLS can be used to measure the sizes of monomeric and aggregated proteins in solution and to deduce the approximate number of monomer units that make up a given state of aggregation. The principle of DLS is based on measuring Brownian motion of proteins (or other macromolecules) when hit by solvent molecules. The detected motion is dependent on molecule size: the larger the molecule, the slower the motion and vice versa. DLS can therefore be used to efficiently deduce the sizes of a mixture of molecules (e.g. a heterogeneous mixture of aggregated
tau proteins), using the hydrodynamic radius which relates to the intensity of molecule fluctuations (Stetefeld et al., 2016).
B A
Figure 1.8. Example data from some commonly used biochemical and biophysical assays for evaluating tau aggregation and conformation.
(A) example standard curve from ThT or ThS kinetic assays (Abedini et al., 2016). (B) Standard CD spectra for proteins which have predominantly a-helices (black), b sheets (red), and unfolded/random coiled (green) conformations. Figure taken from http://www.ap-lab.com/circular_dichroism.htm, accessed on 10th September, 2017.