Conserved and nonconserved features of the folding pathway of hisactophilin, a β‐trefoil protein

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Abstract
Based on previous studies of interleukin-1β (IL-1β) and both acidic and basic fibroblast growth factors (FGFs), it has been suggested that the folding of β-trefoil proteins is intrinsically slow and may occur via the formation of essential intermediates. Using optical and NMR-detected quenched-flow hydrogen/deuterium exchange methods, we have measured the folding kinetics of hisactophilin, another β-trefoil protein that has <10% sequence identity and unrelated function to IL-1β and FGFs. We find that hisactophilin can fold rapidly and with apparently two-state kinetics, except under the most stabilizing conditions investigated where there is evidence for formation of a folding intermediate. The hisactophilin intermediate has significant structural similarities to the IL-1β intermediate that has been observed experimentally and predicted theoretically using a simple, topology-based folding model; however, it appears to be different from the folding intermediate observed experimentally for acidic FGF. For hisactophilin and acidic FGF, intermediates are much less prominent during folding than for IL-1β. Considering the structures of the different β-trefoil proteins, it appears that differences in nonconserved loops and hydrophobic interactions may play an important role in differential stabilization of the intermediates for these proteins.