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.