Removal of the N‐terminal hexapeptide from human β2‐microglobulin facilitates protein aggregation and fibril formation

Abstract

The solution structure and stability of N‐terminally truncated β2‐microglobulin (δN6β2‐m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that δN6β2‐m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full‐length proteins at μM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that δN6β2‐m is significantly less protected than its wild‐type counterpart. Analysis of δN6β2‐m by NMR shows that this loss of protection occurs in β strands I, III, and part of II. At mM concentration gel filtration analysis shows that δN6β2‐m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of β2‐microglobulin was found to have a higher tendency to self‐associate than the intact molecule, and unlike wild‐type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that δN6β2‐m could be a key intermediate of a proteolytic pathway of β2‐microglobulin. Overall, the data suggest that removal of the six residues from the N‐terminus of β2‐microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between β‐strands far removed from this constrain is greatly perturbed.