We generated a panel of single-domain antibodies targeting selected epitopes within tau, using the 'cascade method'. Briefly, we designed complementary peptides targeting linear epitopes within the sequence of tau using a library of beta-sheet fragments from the protein data bank. We then grafted each peptide into the CDR3 loop of a V H human scaffold. Using this approach, we produced a library of fourteen antibodies covering systematically the length of tau. We will use these antibodies to understand which epitopes are specifically exposed in toxic tau aggregates. In particular, we will employ them in superresolved imaging, such as DNA points accumulation for imaging in nanoscale topography (DNA PAINT), of protein aggregates. DNA PAINT enables visualization of structures with resolutions below the diffraction limit using short dye-labelled oligonucleotide probes. Transient binding of a dye-labelled 'imager strand' to a 'docking strand' (DS), which we conjugated to the C-terminus of DesAbs creates a blinking effect. Hence, a highly resolved image can be reconstructed from plotting the measured positions of the hydrolyzed DNA state. Using this technique to image protein aggregates in human cerebrospinal fluid and serum will provide novel insights into the composition, structure, size and number of aggregates present in those samples.

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Spectrin repeat domains are a highly conserved biological motif found in many human structural proteins. Dystrophin contains 24 tandem spectrin repeats which provide structural support through mediating interactions between intracellular actin filaments and the extracellular matrix. However, the molecular mechanism by which dystrophin provides this support is unknown. Understanding this underlying structure/function relationship is important because mutations in dystrophin directly cause muscular dystrophy. Thus far, the following constructs have been expressed in E. coli, purified using chromatography, and thermodynamically characterized: S 17 , S 17-18, S 17-19. Parameters were determined through globally fitting thermal denaturation signals from Fluorescence Spectroscopy (FS), Circular Dichroism (CD), and Fluorescence Lifetime Spectroscopy (FLT) to a two-state model of unfolding. Fits were constrained using DC p values determined using Differential Scanning Calorimetry (DSC). This parameterization then allowed for determination of the free energy of stability (DG unfolding) of each construct. Results indicate that the DG unfolding of S 17 is nearly double that of S 17-19. This pronounced non-additivity indicates that tandem spectrin repeats mutually destabilize each other, termed negative coupling. Additionally, the comparison of Electron Paramagnetic Resonance (EPR) spectra of S 17 and S 17-19 indicate that the trimer exhibits greater local confirmational flexibility, consistent with decreased stability. To further test this negative coupling hypothesis, we are purifying S 19 for thermodynamic characterization. This will allow for the comparison of the sum of S 19 and S 17-18 's DG unfolding values with that of the trimer, helping further reveal the energetic and structural basis of dystrophin's mechanism.

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