Roles for the two N-terminal (β/α) modules in the folding of a (β/α)8-barrel protein as studied by fragmentation analysis
✍ Scribed by Satoshi Akanuma; Akihiko Yamagishi
- Publisher
- John Wiley and Sons
- Year
- 2010
- Tongue
- English
- Weight
- 430 KB
- Volume
- 79
- Category
- Article
- ISSN
- 0887-3585
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✦ Synopsis
Abstract
The (β/α)~8~‐barrel is one of the most abundant folds found in enzymes. To identify the independent folding units and the segment(s) that correspond to a minimum core structure within a (β/α)~8~‐barrel protein, fragmentation experiments were performed with Escherichia coli phosphoribosylanthranilate isomerase, which has a single (β/α)~8~‐barrel domain. Our previous studies indicated that the central four β/α segments comprise an independent folding unit; whereas, the role(s) of the first two β/α segments in folding had not been clarified prior to this report. Herein, we report the design and synthesis of a series of N‐terminally deleted fragments starting with (β/α)~1–5~β~6~ as the parent construct. Analytical gel filtration and urea‐induced equilibrium unfolding experiments indicated that deletions within the N‐terminal region, that is, within the first two β/α modules, resulted in reduced stability or aggregation of the remaining segments. The (β/α)~3–5~β~6~ segment appeared to fold into a stable structure and deletion of β~6~ from (β/α)~3–5~β~6~ yielded (β/α)~3–5~, which did not form native‐like secondary structures. However, urea‐induced unfolding of (β/α)~3–5~, monitored by reduction of tryptophan fluorescence, indicated that the fragment contained a loosely packed hydrophobic core. Taken together, the results of our previous and present fragmentation experiments suggest the importance of the central (β/α)~3–4~β~5~ module in folding, which is a finding that is compatible with our simulated unfolding study performed previously. Proteins 2010. © 2010 Wiley‐Liss, Inc.