pH and urea dependence of amide hydrogen-deuterium exchange rates in the beta-trefoil protein hisactophilin.

Amide hydrogen/deuterium exchange rates were measured as a function of pH and urea for 37 slowly exchanging amides in the beta-trefoil protein hisactophilin. The rank order of exchange rates is generally maintained under different solution conditions, and trends in the pH and urea dependence of exchange rates are correlated with the rank order of exchange rates. The observed trends are consistent with the expected behavior for exchange of different amides via global and/or local unfolding. Analysis of the pH dependence of exchange in terms of rate constants for structural opening and closing reveals a wide range of rates in different parts of the hisactophilin structure. The slowest exchanging amides have the slowest opening and closing rates. Many of the slowest exchanging amides are located in trefoil 2, but there are also some slow exchanging amides in trefoils 1 and 3. Slow exchangers tend to be near the interface between the beta-barrel and the beta-hairpin triplet portions of this single-domain structure. The pattern of exchange behaviour in hisactophilin is similar to that observed previously in interleukin-1 beta, indicating that exchange properties may be conserved among beta-trefoil proteins. Comparisons of opening and closing rates in hisactophilin with rates obtained for other proteins reveal clear trends for opening rates; however, trends in closing rates are less apparent, perhaps due to inaccuracies in the values used for intrinsic exchange rates in the data fitting. On the basis of the pH and urea dependence of exchange rates and optical measurements of stability and folding, EX2 is the main exchange mechanism in hisactophilin, but there is also evidence for varying levels of EX1 exchange at low and high pH and high urea concentrations. Equilibrium intermediates in which subglobal portions of structure are cooperatively disrupted are not apparent from analysis of the urea dependence of exchange rates. There is, however, a strong correlation between the Gibbs free energy of opening and the denaturant dependence of opening for all amides, which suggests exchange from a continuum of states with different levels of structure. Intermediates are not very prominent either in equilibrium exchange experiments or in quenched-flow kinetic studies; hence, hisactophilin may not form partially folded states as readily as IL-1 beta and other beta-trefoil proteins.