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

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)β₆ 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)β₆ segment appeared to fold into a stable structure and deletion of β₆ from (β/α)(3-5)β₆ 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)β₅ module in folding, which is a finding that is compatible with our simulated unfolding study performed previously.