| Project/Area Number |
13480219
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Biophysics
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
GOTO Yuji Osaka University, Institute for Protein Research, Professor, 蛋白質研究所, 教授 (40153770)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
NAIKI Hironobu Fukui University, Medical School, Professor, 医学部, 教授 (10227704)
HOSHINO Masaru Osaka Univ., Inst. for Protein Research, Research Assistant, 蛋白質研究所, 助手 (70304053)
|
|---|
| Project Period (FY) |
2001 – 2003
|
| Project Status |
Completed (Fiscal Year 2003)
|
| Budget Amount *help |
¥14,500,000 (Direct Cost: ¥14,500,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2001: ¥10,900,000 (Direct Cost: ¥10,900,000)
|
|---|
| Keywords | Amyloid fibril / Protein folding / Stability of proteins / Dialysis-related amyloidosis / Fluorescence microscopy / H / D exchange / β2-microglobulin / Amyloid β-peptide / アミロイド病 / 蛋白質 / フォールディング / NMR / 一分子観察 |
|---|
| Research Abstract |
β2-Microglobulin (β2-m)-related amyloidosis is a serious complication in patients receiving long-term hemodialysis. To understand the mechanism of amyloid fibril formation by β2-m, we have been studying the conformation and amyloid fibril formation of recombinant human β2-m.
1.We established a novel procedure using H/D exchange of amide protons combined with NMR analysis for characterizing the conformational flexibility of β2-m amyloid fibrils at single-residue resolution. The results indicated that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid β-sheet core, while the N-and C-termini are not part of this core. The exchange time course deviated largely from a single exponential curve, consistent with the supramolecular structure of fibrils.
2.On the other hand, real-time monitoring of fibril growth is essential to clarify the mechanism of fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. We show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy, amyloid fibrils of β2-m can be visualized without requiring covalent fluorescence labeling. This method was used to follow the kinetics of seed-dependent β2-m fibril extension, revealing the unidirectional extension. Since ThT binding is common to amyloid fibrils, this method will have general applicability as confirmed with the Alzheimer's amyloid β-peptide.
|
