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The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus
1Institute for Protein Research and Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan. yutani@protein.osaka-u.ac.jp
Abstract:
Amyloid is associated with serious diseases including Alzheimer's disease and senile-systemic amyloidosis due to misfolded proteins. In the course of study of the denaturation process of methionine aminopeptidase (MAP) from the hyperthermophile P. furiosus, we found that MAP forms amyloid-like fibrils, and we then investigated the mechanism of amyloid fibril formation. The kinetic experiments on denaturation monitored by CD at 222 nm indicated that MAP in the presence of 3.37 M GuHCl at pH 3.31 changed to a conformation containing a considerable content of beta-sheet structure after the destruction of the alpha-helical structure. MAP in this beta-rich conformation was highly associated, and its stability was remarkably high: the midpoint of the GuHCl denaturation curve was 4.82 M at pH 3.0, and a thermal transition was not observed up to 125 degrees C by calorimetry. The amyloid-like fibril formation of MAP was confirmed by Congo red staining with a typical peak at 542 nm in the difference spectrum, showing a cross-beta X-ray diffraction pattern with a clear sharp reflection at 4.7 A and a characteristic unbranched fibrillar appearance with a length of about 1000 A and a diameter of about 70 A in the electron micrographs. Present results indicate that the amyloid-like form of MAP appears just after the protein is almost completely denatured, and even highly stable proteins can also form amyloid-like conformation under conditions where the denatured state of the protein is abundantly populated.
Insights
Methionine aminopeptidase (MAP) from P. furiosus forms amyloid-like fibrils upon denaturation. This discovery suggests even stable proteins can adopt amyloid conformations under specific denaturing conditions.
Area of Science:
- Biochemistry
- Protein Misfolding Diseases
- Structural Biology
Background:
- Amyloid fibrils are linked to severe diseases like Alzheimer's.
- Protein misfolding and aggregation are key pathological mechanisms.
- Understanding amyloid formation is crucial for therapeutic development.
Purpose of the Study:
- To investigate the mechanism of amyloid fibril formation in methionine aminopeptidase (MAP) from the hyperthermophile P. furiosus.
- To characterize the structural changes and stability of MAP during denaturation and fibril formation.
Main Methods:
- Circular dichroism (CD) spectroscopy to monitor protein denaturation and secondary structure changes.
- Guanidine hydrochloride (GuHCl) denaturation curves to assess protein stability.
- Calorimetry to determine thermal stability.
- Congo red staining and difference spectroscopy to identify amyloid characteristics.
- X-ray diffraction and electron microscopy to analyze fibril structure and morphology.
Main Results:
- MAP forms amyloid-like fibrils under specific denaturing conditions (3.37 M GuHCl, pH 3.31).
- Denaturation involves loss of alpha-helical structure and gain of beta-sheet content.
- The resulting beta-rich conformation exhibits high stability and association.
- Amyloid-like fibrils confirmed by Congo red staining, X-ray diffraction (4.7 A reflection), and electron microscopy (1000 A length, 70 A diameter).
Conclusions:
- Amyloid-like structures form in MAP after significant denaturation.
- Highly stable proteins can form amyloid conformations when their denatured state is populated.
- This finding broadens the understanding of amyloidogenesis beyond typical disease-associated proteins.