Removal of the N-terminal hexapeptide from human beta2-microglobulin facilitates protein aggregation and fibril formation.
The solution structure and stability of N-terminally truncated beta2-microglobulin (deltaN6beta2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that deltaN6beta2-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 microM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that deltaN6beta2-m is significantly less protected than its wild-type counterpart. Analysis of deltaN6beta2-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 deltaN6beta2-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 beta2-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 deltaN6beta2-m could be a key intermediate of a proteolytic pathway of beta2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of beta2-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.
Esposito
G
G
Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, Italy.
Michelutti
R
R
Verdone
G
G
Viglino
P
P
Hernández
H
H
Robinson
C V
CV
Amoresano
A
A
Dal Piaz
F
F
Monti
M
M
Pucci
P
P
Mangione
P
P
Stoppini
M
M
Merlini
G
G
Ferri
G
G
Bellotti
V
V
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D052160:Benzothiazoles; D002850:Chromatography, Gel; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013696:Temperature; D013816:Thermodynamics; D013844:Thiazoles / Q000378:metabolism; D013997:Time Factors; D001613:beta 2-Microglobulin / Q000737:chemistry*
D000682:Amyloid; D052160:Benzothiazoles; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013844:Thiazoles; D001613:beta 2-Microglobulin; C009462:thioflavin T