The role of disulfide bond in the amyloidogenic state of beta(2)-microglobulin studied by heteronuclear NMR.
beta(2)-Microglobulin (beta2-m) is a major component of dialysis-related amyloid fibrils. Although recombinant beta2-m forms needle-like fibrils by in vitro extension reaction at pH 2.5, reduced beta2-m, in which the intrachain disulfide bond is reduced, cannot form typical fibrils. Instead, thinner and flexible filaments are formed, as shown by atomic force microscopy images. To clarify the role of the disulfide bond in amyloid fibril formation, we characterized the conformations of the oxidized (intact) and reduced forms of beta2-m in the acid-denatured state at pH 2.5, as well as the native state at pH 6.5, by heteronuclear NMR. [(1)H]-(15)N NOE at the regions between the two cysteine residues (Cys25-Cys80) revealed a marked difference in the pico- and nanosecond time scale dynamics between that the acid-denatured oxidized and reduced states, with the former showing reduced mobility. Intriguingly, the secondary chemical shifts, DeltaCalpha, DeltaCO, and DeltaHalpha, and (3)J(HNHalpha) coupling constants indicated that both the oxidized and reduced beta2-m at pH 2.5 have marginal alpha-helical propensity at regions close to the C-terminal cysteine, although it is a beta-sheet protein in the native state. The results suggest that the reduced mobility of the denatured state is an important factor for the amylodogenic potential of beta2-m, and that the marginal helical propensity at the C-terminal regions might play a role in modifying this potential.
Katou
Hidenori
H
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Kanno
Takashi
T
Hoshino
Masaru
M
Hagihara
Yoshihisa
Y
Tanaka
Hiroyuki
H
Kawai
Tomoji
T
Hasegawa
Kazuhiro
K
Naiki
Hironobu
H
Goto
Yuji
Y
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000235:genetics
D000682:Amyloid; D004220:Disulfides; D009587:Nitrogen Isotopes; D001613:beta 2-Microglobulin; D006859:Hydrogen