The role of the I(T)-state in D76N β(2)-microglobulin amyloid assembly: A crucial intermediate or an innocuous bystander?

The D76N variant of human β(2)-microglobulin (β(2)m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of WT-β(2)m, which occurs in dialysis-related amyloidosis. A folding intermediate of WT-β(2)m, known as the I(T)-state, which contains a nonnative trans Pro-32, has been shown to be a key precursor of WT-β(2)m aggregation in vitro. However, how a single amino acid substitution enhances the rate of aggregation of D76N-β(2)m and gives rise to a different amyloid disease remained unclear. Using real-time refolding experiments monitored by CD and NMR, we show that the folding mechanisms of WT- and D76N-β(2)m are conserved in that both proteins fold slowly via an I(T)-state that has similar structural properties. Surprisingly, however, direct measurement of the equilibrium population of I(T) using NMR showed no evidence for an increased population of the I(T)-state for D76N-β(2)m, ruling out previous models suggesting that this could explain its enhanced aggregation propensity. Producing a kinetically trapped analog of I(T) by deleting the N-terminal six amino acids increases the aggregation rate of WT-β(2)m but slows aggregation of D76N-β(2)m, supporting the view that although the folding mechanisms of the two proteins are conserved, their aggregation mechanisms differ. The results exclude the I(T)-state as the origin of the rapid aggregation of D76N-β(2)m, suggesting that other nonnative states must cause its high aggregation rate. The results highlight how a single substitution at a solvent-exposed site can affect the mechanism of aggregation and the resulting disease.