Dynamic structures of rare conformations involved in amyloid formation

In work described in this thesis the conformational landscape of a recently identified ß2m variant, D76N, was examined with NMR and state of the art molecular dynamics simulations. The D76N ß2m variant was recently identified as the first naturally occurring amyloidogenic variant of ß2m, a 99 residue, seven stranded Ig domain protein. The D76N variant of ß2m causes a hereditary, late onset and fatal systemic amyloid disease. Affected individuals are heterozygous for the D76N mutation and thus also express wild-type (WT) ß2m in the serum. Co-aggregation of D76N and WT ß2m is not observed in vivo, and D76N amyloidosis follows a distinctive disease progression from the WT ß2m associated disease, Dialysis-Related Amyloidosis, which occurs in patients undergoing long-term haemodialysis. This work aims to understand the mechanism by which the single amino acid substitution, D76N, results in a dramatically more aggregation-prone protein while maintaining the same overall native structure as WT ß2m. In this work, the structure, stability and dynamics of the D76N variant are studied with NMR spectroscopy and a range of complementary biophysical techniques. The folding intermediates of D76N and WT ß2m are also probed with real-time NMR and circular dichroism spectroscopy, revealing structural, dynamic and kinetic insights into the folding intermediates present within the D76N and WT ß2m folding pathways and their roles in aggregation. The study of the folding intermediates demonstrated that the IT -state, which is a known precursor for ß2m aggregation, is not responsible for the rapid aggregation of the D76N variant. Instead, a low-populated state, unique to D76N, in equilibrium with the native state must be responsible for D76N aggregation, consistent with the different disease progression associated with D76N. The NMR and data-guided molecular dynamics simulations presented in this thesis reveal the effects of the D76N substitution on the conformational landscape of the D76N variant and detail the structures of the low-populated states in equilibrium with the native state. Additionally, saturation mutagenesis of position 76 in D76N, carried out with a split-ßlactamase assay have demonstrated the exquisite specificity for an aspartate residue at position 76 in the ß2m structural ensemble.