The mechanism of the amyloidogenic conversion of T7 endonuclease I.
Amyloid fibrils are associated with a range of human disorders. Understanding the conversion of amyloidogenic proteins from their soluble forms to amyloid fibrils is critical for developing effective therapeutics. Previously we showed that T7 endonuclease I forms amyloid-like fibrils. Here we study the mechanism of the amyloidogenic conversion of T7 endonuclease I. We show that T7 endonuclease I forms fibrils at pH 6.8, but not at pH 6.0 or 8.0. The amyloidogenicity at pH 6.8 is not correlated with thermodynamic stability, unfolding cooperativity, or solubility. Thermal melting experiments at various pH values show that the protein has a distinctive thermal transition at pH 6.8. The transition at pH 6.8 has a lower transition temperature than the unfolding transitions observed at pH 6.0 and 8.0 and leads to a beta-rich conformation instead of an unfolded state. Electron microscopy shows that the thermal transition at pH 6.8 results in fibril formation. The thermal transition at pH 6.8 leads to a protein state that is not accessible at pH 6.0 or 8.0, showing that the existence of the amyloidogenic conformation of T7 endonuclease I depends sensitively on solution conditions. Therefore, we propose that fibrillizing proteins need to be "prepared" for fibrillization. Preparation may consist of amino acid replacements or changing solution conditions and may require retention of some aspects of native structure. In this model, some amyloid-enhancing mutations decrease protein stability, whereas others have little effect.
Guo
Zhefeng
Z
Howard Hughes Medical Institute, UCLA-Department of Energy Institute for Genomics and Proteomics, Molecular Biology Institute, UCLA, Los Angeles, California 90095-1570, USA.
Eisenberg
David
D
D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D017123:Bacteriophage T7 / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D020125:Mutation, Missense; D017510:Protein Folding; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
D000682:Amyloid; D014764:Viral Proteins; D003850:Deoxyribonuclease I