Amyloid fibril formation in the context of full-length protein: effects of proline mutations on the amyloid fibril formation of beta2-microglobulin.
Beta2-microglobulin (beta2-m), a typical immunoglobulin domain made of seven beta-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis. To understand the mechanism of amyloid fibril formation in the context of full-length protein, we prepared various mutants in which proline (Pro) was introduced to each of the seven beta-strands of beta2-m. The mutations affected the amyloidogenic potential of beta2-m to various degrees. In particular, the L23P, H51P, and V82P mutations significantly retarded fibril extension at pH 2.5. Among these, only L23P is included in the known "minimal" peptide sequence, which can form amyloid fibrils when isolated as a short peptide. This indicates that the residues in regions other than the minimal sequence, such as H51P and V82P, determine the amyloidogenic potential in the full-length protein. To further clarify the mutational effects, we measured their stability against guanidine hydrochloride of the native state at pH 8.0 and the amyloid fibrils at pH 2.5. The amyloidogenicity of mutants showed a significant correlation with the stability of the amyloid fibrils, and little correlation was observed with that of the native state. It has been proposed that the stability of the native state and the unfolding rate to the amyloidogenic precursor as well as the conformational preference of the denatured state determine the amyloidogenicity of the proteins. The present results reveal that, in addition, stability of the amyloid fibrils is a key factor determining the amyloidogenic potential of the proteins.
Chiba
Takeshi
T
Institute for Protein Research, Osaka University, and CREST, Japan Science and Technology Corp., Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Hagihara
Yoshihisa
Y
Higurashi
Takashi
T
Hasegawa
Kazuhiro
K
Naiki
Hironobu
H
Goto
Yuji
Y
D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D019281:Dimerization; D019791:Guanidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011392:Proline; D011489:Protein Denaturation; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000648:ultrastructure
D000682:Amyloid; D001613:beta 2-Microglobulin; D011392:Proline; D019791:Guanidine