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Roles for the two N-terminal (β/α) modules in the folding of a (β/α)8-barrel protein as studied by fragmentation analysis

Authors

  • Satoshi Akanuma,

    1. Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
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  • Akihiko Yamagishi

    Corresponding author
    1. Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
    • Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
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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)1–5β6 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)3–5β6 segment appeared to fold into a stable structure and deletion of β6 from (β/α)3–5β6 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)3–4β5 module in folding, which is a finding that is compatible with our simulated unfolding study performed previously. Proteins 2010. © 2010 Wiley-Liss, Inc.

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