Native-unlike long-lived intermediates along the folding pathway of the amyloidogenic protein beta2-microglobulin revealed by real-time two-dimensional NMR.
Beta2-microglobulin (beta2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified beta2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G beta2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G beta2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the beta2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type beta2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G beta2m mutant.
Corazza
Alessandra
A
Department of Biomedical Science and Technology, University of Udine, Piazzale Kolbe 4, 33100 Udine, Italy. alessandra.corazza@uniud.it
Rennella
Enrico
E
Schanda
Paul
P
Mimmi
Maria Chiara
MC
Cutuil
Thomas
T
Raimondi
Sara
S
Giorgetti
Sofia
S
Fogolari
Federico
F
Viglino
Paolo
P
Frydman
Lucio
L
Gal
Maayan
M
Bellotti
Vittorio
V
Brutscher
Bernhard
B
Esposito
Gennaro
G
D019943:Amino Acid Substitution; D000682:Amyloid / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000235:genetics
D000682:Amyloid; D001613:beta 2-Microglobulin