The formation of partially unfolded intermediates through conformational excursions out of the native state is the starting point of many diseases involving protein aggregation. Therapeutic strategies often aim to stabilize the native structure and prevent the formation of intermediates that are also cytotoxic in vivo. However, their transient nature and low population makes it difficult to characterize these intermediates. We have probed the backbone dynamics of transthyretin (TTR) over an extended timescale by using NMR spectroscopy and MD simulations. The location and extent of these motions indicates that the backbone flexibility of TTR is a cause of dissociation and destabilization, both of which are responsible for fibril formation. Importantly, approximately 10 % of wild-type TTR exists in an intermediate state, which increased to up to 28 % for pathogenic TTR mutants, for which the formation of the intermediate state is shown to be energetically more favorable compared to the wild type. This result suggests an important role for the intermediates in TTR amyloidosis.

The formation of partially unfolded intermediates through conformational excursions out of the native state is the starting point of many diseases involving protein aggregation. Therapeutic strategies often aim to stabilize the native structure and prevent the formation of intermediates that are also cytotoxic in vivo. However, their transient nature and low population makes it difficult to characterize these intermediates. We have probed the backbone dynamics of transthyretin (TTR) over an extended timescale by using NMR spectroscopy and MD simulations. The location and extent of these motions indicates that the backbone flexibility of TTR is a cause of dissociation and destabilization, both of which are responsible for fibril formation. Importantly, approximately 10 % of wild‐type TTR exists in an intermediate state, which increased to up to 28 % for pathogenic TTR mutants, for which the formation of the intermediate state is shown to be energetically more favorable compared to the wild type. This result suggests an important role for the intermediates in TTR amyloidosis.

Immunoglobulin light chains (LCs) deposit as insoluble aggregates causing a disease called light chain amyloidosis (AL) [1]. Among the LC families, lambda 6a is very frequent in AL patients. Its germline protein (6aJL2) and point mutants (R24G, P7S and D52A) are good models to study fibrilllogenesis, because these mutations have drastic effects in native state stability and in fibril formation [2,3]. As yet, the conformational changes resulting in LC intermediates able to form fibrils have not been characterized at the atomic level. At the very least, these changes should involve eliminating the anti-aggregation motifs [4] and rotating the b sheets so the strands become parallel [5]. From high temperature molecular dynamic simulations of these proteins, we identified unfolding intermediates filling the above criteria. The chosen structures retain native conformation, while the sides of the beta sandwich remain denatured. We quenched them at 298 K and performed molecular dynamics simulations (10 replicates x 10 ns each). Interestingly all the intermediates held their structured core within simulation time, with an RMSD from the initial structure below 2.5 Å. Based on the hypothesis that early denaturation states of these proteins can lead to amyloid aggregation, we propose that these intermediates could fit in the amyloid core, and they could be stable enough to initiate the aggregation process.