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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.

Original publication

DOI

10.1110/ps.9.5.831

Type

Journal article

Journal

Protein Sci

Publication Date

05/2000

Volume

9

Pages

831 - 845

Keywords

Amino Acid Sequence, Amyloid, Chromatography, Gel, Circular Dichroism, DNA, Complementary, Humans, Hydrogen Bonding, Hydrogen-Ion Concentration, Light, Magnetic Resonance Spectroscopy, Mass Spectrometry, Models, Molecular, Molecular Sequence Data, Protein Conformation, Protein Structure, Secondary, Recombinant Proteins, Scattering, Radiation, Temperature, Thermodynamics, Thiazoles, Time Factors, beta 2-Microglobulin