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

The (δ/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 (δ/α) ₈-barrel protein, fragmentation experiments were performed with Escherichia coli phosphoribosylanthranilate isomerase, which has a single (δ/α) ₈-barrel domain. Our previous studies indicated that the central four δ/α segments comprise an independent folding unit; whereas, the role(s) of the first two δ/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 (δ/α) _1-5δ 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 δ/α modules, resulted in reduced stability or aggregation of the remaining segments. The (δ/α) _3-5δ ₆ segment appeared to fold into a stable structure and deletion of δ ₆ from (δ/α) _3-5δ ₆ yielded (δ/α) _3-5, which did not form native-like secondary structures. However, urea-induced unfolding of (δ/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 (δ/α) _3-4δ ₅ module in folding, which is a finding that is compatible with our simulated unfolding study performed previously. Proteins 2010.