Folding mechanisms of a naturally evolved and two artificial (beta alpha) (8)-barrels: a comparative analysis

We compared the stability and the folding kinetics of the natively evolved (βα)₈-barrel enzyme HisF (imidazole glycerol phosphate synthase) and of the artificial (βα)₈-barrels HisF-C***C and HisF-sym, which were designed by fusing and stabilizing identical (βα)₄ half-barrels. HisF-C***C is less stable, HisF-sym is more stable than HisF, but all three proteins unfold with similar cooperativity in denaturant-induced transitions. Fundamental characteristics are identical in the folding pathways of the two artificial and the naturally evolved protein: The folding rates as determined by Trp fluorescence and far-UV CD changes coincide for the individual proteins, indicating that secondary and tertiary structures are formed and unfolded simultaneously. Above 2 M GdmCl, unfolding and refolding are single-exponential reactions for HisF as well as for HisF-C***C, and HisF-sym. Below 2 M GdmCl, refolding is complex. For all three proteins, HisF, HisF-C***C, and HisF-sym, a burst-phase folding intermediate is observed by far-UV CD, followed by the formation of a further folding intermediate. Its conversion to the native state is the rate-limiting step in the folding reactions of all three proteins. Thus, the folding pathways of HisF, HisF-C***C and HisF-sym show similarities which further supports the hypothesis that the (βα)₈-barrel fold has evolved by duplication and fusion of (βα)₄ half-barrels.