Removal of the N-terminal hexapeptide from human beta 2-microglobulin facilitates protein aggregation and fibril formation

The solution structure and stability of N-terminally truncated beta 2-micro globulin (Delta N6 beta 2-m), the major modification in ex vivo fibrils, ha ve been investigated by a variety of biophysical techniques. The results sh ow that Delta N6 beta 2-m has a free energy of stabilization that is reduce d by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mi xture of the truncated and full-length proteins at mu M concentrations at p H 6.5 monitored by electrospray mass spectrometry reveals that Delta N6 bet a 2-m is significantly less protected than its wild-type counterpart. Analy sis of Delta N6 beta 2-m by NMR shows that this loss of protection occurs i n beta strands I, III, and part of II. At mM concentration gel filtration a nalysis shows that Delta N6 beta 2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involve d in intermolecular interactions that stabilize this association. The trunc ated 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 exper iments and analysis by mass spectrometry support the conformational modific ations 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. Pa rt of the tertiary structure is preserved substantially by the disulfide br idge between Cys25 and Cys80, but the pairing between beta-strands far remo ved from this constrain is greatly perturbed.