Amyloid formation is implicated in a number of human diseases. β2-microglobulin (β2m) is the precursor protein in dialysis-related amyloidosis and it has been shown that partial, or more complete, unfolding is key to amyloid fibril formation in this pathology. Here the relationship between conformational flexibility and β2m amyloid formation at physiological pH has been investigated.
HDX-ESI-MS was used to study the conformational dynamics of β2m. Protein engineering, or the addition of Cu2+ ions, sodium dodecyl sulphate, trifluoroethanol, heparin, or protein stabilisers, was employed to perturb the conformational dynamics of β2m. The fibril-forming propensities of the protein variants and the wild-type protein in the presence of additives, which resulted in >5-fold increase in the EX1 rate of HDX, were investigated further.
ESI-MS revealed that HDX occurs via a mixed EX1/EX2 mechanism under all conditions. Urea denaturation and tryptophan fluorescence indicated that EX1 exchange occurred from a globally unfolded state in wild-type β2m. Although >30-fold increase in the HDX exchange rate was observed both for the protein variants and for the wild-type protein in the presence of specific additives, large increases in exchange rate did not necessarily result in extensive de novo fibril formation.
The conformational dynamics measured by the EX1 rate of HDX do not predict the ability of β2m to form amyloid fibrils de novo at neutral pH. This suggests that the formation of amyloid fibrils from β2m at neutral pH is dependent on the generation of one or more specific aggregation-competent species which facilitate self-assembly. Copyright © 2012 John Wiley & Sons, Ltd.