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

The solution structure and stability of N-terminally truncated beta 2-microglobulin (Delta N6 beta 2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that Delta N6 beta 2-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 mu M concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that Delta N6 beta 2-m is significantly less protected than its wild-type counterpart. Analysis of Delta N6 beta 2-m by NMR shows that this loss of protection occurs in beta strands I, III, and part of II. At mM concentration gel filtration analysis shows that Delta N6 beta 2-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 beta 2-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 Delta N6 beta 2-m could be a key intermediate of a proteolytic pathway of beta 2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of beta 2-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 beta-strands far removed from this constrain is greatly perturbed.