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.

Based on previous studies of interleukin-1␤ (IL-1␤) and both acidic and basic fibroblast growth factors (FGFs), it has been suggested that the folding of ␤-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 ␤-trefoil protein that has <10% sequence identity and unrelated function to IL-1␤ 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-1␤ 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-1␤. Considering the structures of the different ␤-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.

Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/ refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (Յ18 kD) that are normally soluble in vivo. In addition, refolding the purified proteins by using dialysis against a single buffer allowed us to obtain soluble protein samples of 58% of small proteins that were found in the insoluble fraction in vivo, and 10% of the initial number of proteins provided good heteronuclear single quantum coherence (HSQC) NMR spectra. We conclude that a denaturation/refolding protocol is an efficient way to generate structural samples for high-throughput studies of small proteins.Keywords: Protein structure/folding; protein solubility; protein purification; NMR spectroscopy; circular dichroism spectroscopy; structural proteomics Structure determination using NMR spectroscopy and X-ray crystallography requires the generation of large amounts of soluble recombinant protein. Most often, recombinant proteins for structural biology studies are produced in Escherichia coli because the cells grow rapidly and to high density in inexpensive medium, the expression system is well characterized, and a large number of expression vector systems and mutant host strains are available. In addition, under appropriate conditions, the recombinant protein can comprise >50% of the total cellular protein. However, the use of the E. coli expression system is limited because the target proteins often segregate partially or completely into the insoluble fraction of the cell. Recent studies of protein expression on a large number of prokaryotic and eukaryotic proteins indicate that >50% of recombinant proteins may be found in the insoluble fraction of bacterial cell lysates (Christendat et al. 2000a,b;Yee et al. 2002).Several strategies have been used to increase the probability of producing soluble recombinant proteins in bacterial cells, including co-expressing protein folding modulators, manipulating the temperature of growth and induction, and producing the protein as a fusion with another soluble Reprint requests to: Aled M. Edwards, Banting and Best

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