Abstract
AAI, the major alpha-amylase inhibitor (AAI) present in the seeds of the Mexican crop plant Amaranthus hypocondriacus is a 32-residue-long polypeptide with three disulfide bridges. Its structure is most closely related to the plant amylase inhibitor subfamily of knottins characterized by a topological knot formed by one disulfide bridge threading through a loop formed by the peptide chain as well as a short three-stranded beta sandwich core. AAI is specific against insect amylases and does not act on corresponding human or mammalian enzymes. It was found that the oxidative folding of AAI seems to follow a hirudine-like pathway with many non-native intermediates, but notably it proceeds through a major folding intermediate (MFI) that contains a vicinal disulfide bridge. Based on a review of the pertinent literature, the known vicinal disulfides in native proteins as well as well as the network of disulfide interchanges, we propose that MFI is a kinetic trap corresponding to a compact molten globule-like state which constrains the peptide chain to a smaller number of conformations that in turn can be rapidly funneled toward the native state.
Highlights
SYNTHESIS AND IN VITRO OXIDATIVE FOLDING OF AAIThe AAI peptide was synthesized manually on a 1-mmol scale by solid-phase peptide synthesis using an Fmoc (N(9-fluorenyl)methoxycarbonyl) methodology (Lozanov et al, 1997; Cemazar et al, 2003)
We propose that future protein design attempts could make use of the large interaction surface of AAI as well as its unique folding mechanism to explore novel possible applications and synthetic routes
Summary
The AAI peptide was synthesized manually on a 1-mmol scale by solid-phase peptide synthesis using an Fmoc (N(9-fluorenyl)methoxycarbonyl) methodology (Lozanov et al, 1997; Cemazar et al, 2003). Even though native AAI and MFI possess different disulfide pairings, their sizes are indistinguishable within the experimental error (Cemazar et al, 2004) This makes us believe that MFI may play a role similar to that of the molten globule state of larger proteins by constraining the peptide chain to a smaller number of conformations that can be rapidly funneled toward the native state. In order to get insights into the role the intermediates we prepared a folding map with intermediates as the nodes and disulfide exchange reactions as the edges (Agoston et al, 2005) Time resolved NMR revealed a monotonous change in the aliphatic and aromatic NMR signals, respectively, and confirmed that enzyme catalyzed reaction was somewhat faster, even though it proceeded through the same intermediates, with minor quantitative differences at the first stages of the reaction (Carugo et al, 2003)
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