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

Gennaro Esposito, R. Michelutti, G. Verdone, P. Viglino, H. Hernández, C. V. Robinson, A. Amoresano, F. Dal Piaz, M. Monti, P. Pucci, P. Mangione, M. Stoppini, G. Merlini, G. Ferri, Vittorio Bellotti

Research output: Contribution to journalArticle

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 study 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.

Original languageEnglish (US)
Pages (from-to)831-845
Number of pages15
JournalProtein Science
Volume9
Issue number5
DOIs
StatePublished - Jan 1 2000

Fingerprint

Agglomeration
Nuclear magnetic resonance
Mass spectrometry
Mass Spectrometry
Proteolysis
Proteins
Oligomers
Amyloid
Disulfides
Free energy
Gel Chromatography
Hydrogen
Stabilization
Gels
Association reactions
Molecules
Experiments

Keywords

  • Amyloidosis
  • Hydrogen exchange mass spectrometry
  • Limited proteolysis
  • NMR
  • Protein folding
  • β2-microglobulin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Esposito, G., Michelutti, R., Verdone, G., Viglino, P., Hernández, H., Robinson, C. V., ... Bellotti, V. (2000). Removal of the N-terminal hexapeptide from human β2-microglobulin facilities protein aggregation and fibril formation. Protein Science, 9(5), 831-845. https://doi.org/10.1110/ps.9.5.831

Removal of the N-terminal hexapeptide from human β2-microglobulin facilities protein aggregation and fibril formation. / Esposito, Gennaro; Michelutti, R.; Verdone, G.; Viglino, P.; Hernández, H.; Robinson, C. V.; Amoresano, A.; Dal Piaz, F.; Monti, M.; Pucci, P.; Mangione, P.; Stoppini, M.; Merlini, G.; Ferri, G.; Bellotti, Vittorio.

In: Protein Science, Vol. 9, No. 5, 01.01.2000, p. 831-845.

Research output: Contribution to journalArticle

Esposito, G, Michelutti, R, Verdone, G, Viglino, P, Hernández, H, Robinson, CV, Amoresano, A, Dal Piaz, F, Monti, M, Pucci, P, Mangione, P, Stoppini, M, Merlini, G, Ferri, G & Bellotti, V 2000, 'Removal of the N-terminal hexapeptide from human β2-microglobulin facilities protein aggregation and fibril formation', Protein Science, vol. 9, no. 5, pp. 831-845. https://doi.org/10.1110/ps.9.5.831
Esposito, Gennaro ; Michelutti, R. ; Verdone, G. ; Viglino, P. ; Hernández, H. ; Robinson, C. V. ; Amoresano, A. ; Dal Piaz, F. ; Monti, M. ; Pucci, P. ; Mangione, P. ; Stoppini, M. ; Merlini, G. ; Ferri, G. ; Bellotti, Vittorio. / Removal of the N-terminal hexapeptide from human β2-microglobulin facilities protein aggregation and fibril formation. In: Protein Science. 2000 ; Vol. 9, No. 5. pp. 831-845.
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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 study 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.",
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AU - Verdone, G.

AU - Viglino, P.

AU - Hernández, H.

AU - Robinson, C. V.

AU - Amoresano, A.

AU - Dal Piaz, F.

AU - Monti, M.

AU - Pucci, P.

AU - Mangione, P.

AU - Stoppini, M.

AU - Merlini, G.

AU - Ferri, G.

AU - Bellotti, Vittorio

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