Conformation of Amyloid Fibrils of β2-Microglobulin Probed by Tryptophan Mutagenesis

Miho Kihara,Eri Chatani,Kentaro Iwata,Kaori Yamamoto,Takanori Matsuura,Atsushi Nakagawa,Hironobu Naiki,Yuji Goto

doi:10.1074/jbc.m605358200

Miho Kihara, Eri Chatani + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m605358200

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Abstract

Beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, adopts an immunoglobulin domain fold in its native state. Although beta2-m has Trp residues at positions 60 and 95, both are located near the surface of the domain. Hence, beta2-m does not have a conserved Trp common to other immunoglobulin domains, which is buried in close proximity to the disulfide bond. To study the structure of amyloid fibrils in relation to their native fold, we prepared a series of Trp mutants. Trp60 and Trp95 were both replaced with Phe, and a single Trp was introduced at various positions. Among various mutants, W39-beta2-m, in which a Trp was introduced at the position corresponding to the conserved Trp, exhibited a remarkable quenching of fluorescence in the native state, as observed for other immunoglobulin domains. An x-ray structural analysis revealed that W39-beta2-m assumes the native fold with Trp39 located in the vicinity of the disulfide bond. Comparison of the fluorescence spectra of various mutants for the native and fibrillar forms indicated that, while the Trp residues introduced in the middle of the beta2-m sequence tend to be buried in the fibrils, those located in the C-terminal region are more exposed. In addition, the fluorescence spectra of fibrils prepared at pH 2.5 and 7.0 revealed a large difference in the fluorescence intensity for W60-beta2-m, implying a major structural difference between them.

Highlights

Size for examining the relation between protein folding and amyloid fibril formation [4,5,6,7,8,9,10,11]. ␤2-m, a typical immunoglobulin domain made of seven ␤-strands and one intramolecular disulfide bond [12, 13], is present as the non-polymorphic light chain of the class I major histocompatibility complex [14] (Fig. 1)
The near-UV CD spectra were differed, indicating that Trp residues contribute to the near-UV CD and that the environment of these residues differs depending on the mutant
For most of the single Trp mutants, we observed signal changes indicating a significant conformational change between the native structure and amyloid fibrils. This suggests that the formation of amyloid fibrils of ␤2-m accompanies a significant conformational change to the entire molecule

Summary

Introduction

Size for examining the relation between protein folding and amyloid fibril formation [4,5,6,7,8,9,10,11]. ␤2-m, a typical immunoglobulin domain made of seven ␤-strands and one intramolecular disulfide bond [12, 13], is present as the non-polymorphic light chain of the class I major histocompatibility complex [14] (Fig. 1). ␤2-m Amyloid Fibril Formation—The expression and purification of human recombinant ␤2-m and the single Trp mutants were achieved as described previously [21]. Acidic pH fibrils were prepared by a repeated seed-dependent extension with human recombinant ␤2-m expressed in Escherichia coli [21]. The amyloid extension reactions of mutant ␤2-ms were carried out using the wild-type neutral pH seeds and wild-type acidic pH seeds at pH 7.0 and 2.5, respectively. The extension reaction mixture at pH 2.5 contained 25 ␮M monomeric ␤2-m and 5 ␮g/ml acidic seeds. The extension reaction mixture at pH 7.0 contained 25 ␮M monomeric ␤2-m, 30 ␮g/ml neutral pH seeds, and 0.5 mM SDS [23]. The reaction was monitored by the fluorometric assay with thioflavin T (ThT) at pH 8.5 as described previously [21,22,23]

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