The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus.
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.
Yutani
K
K
Institute for Protein Research and Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan. yutani@protein.osaka-u.ac.jp
Takayama
G
G
Goda
S
S
Yamagata
Y
Y
Maki
S
S
Namba
K
K
Tsunasawa
S
S
Ogasahara
K
K
D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D063208:Methionyl Aminopeptidases; D011489:Protein Denaturation / Q000187:drug effects; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000378:metabolism; D014461:Ultracentrifugation
D000682:Amyloid; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; D019791:Guanidine