Molecular level insights into thermally induced α-chymotrypsinogen A amyloid aggregation mechanism and semiflexible protofibril morphology.
Understanding nonnative protein aggregation is critical not only to a number of amyloidosis disorders but also for the development of effective and safe biopharmaceuticals. In a series of previous studies [Weiss et al. (2007) Biophys. J. 93, 4392-4403; Andrews et al. (2007) Biochemistry 46, 7558-7571; Andrews et al. (2008) Biochemistry 47, 2397-2403], α-chymotrypsinogen A (aCgn) and bovine granulocyte colony stimulating factor (bG-CSF) have been shown to exhibit the kinetic and morphological features of other nonnative aggregating proteins at low pH and ionic strength. In this study, we investigated the structural mechanism of aCgn aggregation. The resultant aCgn aggregates were found to be soluble and exhibited semiflexible filamentous aggregate morphology under transmission electron microscopy. In addition, the filamentous aggregates were demonstrated to possess amyloid characteristics by both Congo red binding and X-ray diffraction. Peptide level hydrogen exchange (HX) analysis suggested that a buried native β-sheet comprised of three peptide segments (39-46, 51-64, and 106-114) reorganizes into the cross-β amyloid core of aCgn aggregates and that at least ∼50% of the sequence adopts a disordered structure in the aggregates. Furthermore, the equimolar, bimodal HX labeling distribution observed for three reported peptides (65-102, 160-180, and 229-245) suggested a heterogeneous assembly of two molecular conformations in aCgn aggregates. This demonstrates that extended β-sheet interactions typical of the amyloid are sufficiently strong that a relatively small fraction of polypeptide sequence can drive formation of filamentous aggregates even under conditions favoring colloidal stability.
Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904, United States.
Aming
A
Zhang
Jacob L
JL
Jordan
Magdalena I
MI
Ivanova
William F
WF
Weiss
Christopher J
CJ
Roberts
Erik J
EJ
Fernandez
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism*; D000686:Amyloidosis / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002919:Chymotrypsinogen / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D003224:Congo Red / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D018583:Pliability; D014961:X-Ray Diffraction
D000682:Amyloid; D003224:Congo Red; D002919:Chymotrypsinogen