Modulators of Protein Folding and Misfolding

The folded structure of a protein allows for its biological function. Though proteins may adopt a folded structure, they can also misfold into stable conformations that result in loss of physiological function. The structure of a peptide is dependent upon the amino acid sequence, the interactions within the peptide chain, and environmental factors. Therefore, peptides derived from a portion of a particular protein structure may not adopt the fold of the parent sequence although they contain the same residues. Peptidomimetic strategies to stabilize specific conformations typically rely on alteration of the peptide sequence. These modifications include selection of specific side chains, macrocyclization, and backbone modification. Peptidomimetics that modulate the stability of folded and misfolded conformations can be used as therapeutics and tool molecules. Misfolding of the tau protein is correlated with the progression of several neurodegenerative diseases. In this work, we utilized careful side chain selection and peptide stapling techniques to afford cross-beta epitope mimics of conformation strains of misfolded tau. We demonstrated through a series of biochemical and biophysical experiments that these mimics, which we call beta-bracelets, self-assemble into cross-beta fibrils similar to those of misfolded tau. Installation of an N-amino substituent onto the backbone amide of beta-bracelet tau mimics precludes self-assembly and converts amyloidogenic macrocycles into inhibitors of tau aggregation. The propagation of misfolded tau fibrils is central to the progression of tauopathies. We further show in cell-based assays that N-aminated beta-bracelets are effective inhibitors of propagation induced by tau seeds derived from recombinant protein or patient brain extracts. This work marks an important step in beta-arch mimicry and peptide inhibitors of tau aggregation and propagation. Tau aggregates adopt unique structures based on the disease they are associated with. Currently, model peptides that adopt or induce pathological tau folds are lacking. Continuing our pursuit of tau-derived peptidomimetics, we applied the same macrocyclic stapling strategy to mimic beta-arch structures from the cores of pathological tau folds. We demonstrate that our beta-arch mimics self-assemble into stable beta-sheet fibrils that show cross-beta diffraction patterns. Further, these minimal tau macrocycles are capable of inducing tau aggregation in bioengineered cell lines and primary mouse neurons. Cryo-EM reconstruction of filaments formed by these compounds reveals conformational features present in various 4R tauopathic folds. This work identifies beta-arch cores of 4R tauopathies as important for disease-associated tau propagation and may lead to the development of conformational antibodies and tau targeted vaccines. C(alpha)-substituted alpha-hydrazino acids stabilize beta-sheet conformation, however, the effect of N-aminoglycine (aGly) is unexplored. We investigated the effect of aGly substitution on polyproline II (PPII) secondary structure. A considerable portion of residues in known protein structures are found within PPII helices. Additionally, PPII secondary structure is important for mediating protein-protein interactions involving SH3 domains. Incorporation of various N-aminated residues in a PPII octapeptide model system revealed that aGly enhanced PPII helicity through increased electron delocalization interactions. Further, we utilized the reactivity of backbone N-amino groups to afford alkylated aGly derivatives. Two of these derivatives enhanced PPII helicity beyond that of aGly. Additionally, variable temperature circular dichroism experiments revealed increased thermal stability of aGly and isopropyl-aGly containing PPII helices. The trend of increased thermal stability of alkylated aGly derivatives was demonstrated upon incorporation of these residues in the PPII-containing avian pancreatic polypeptide. We believe that this work may inform the rational design of stabilized PPII peptidomimetics as modulators of protein-protein interactions.