Intramolecular Cross-Linking Evaluated as a Structural Probe of the Protein Folding Transition State†

A continuing goal of experimental protein folding studies is to elucidate the structure of the transition state ensemble (TSE) in the hopes of understanding the critical steps of the folding process. To identify regions structurally important in the TSE, many studies observe the changes in kinetic response that result from a perturbation. In mutational ϕ-analysis, the perturbation takes the form of point mutations engineered throughout the protein (1–3). The accompanying changes in folding rates reports on the mutated side-chain’s level of energetic involvement in the TS. The extent of involvement is quantified as the ϕ-value, defined as the change in folding activation energy relative to the change in equilibrium stability, ϕ = ΔΔG‡/ ΔΔGeq. A new method, ψ-analysis, employs metal ion binding to engineered bihistidine (biHis) sites as the means of perturbation (4–9) (Fig. 1). An increase in divalent metal ion concentration stabilizes the interaction between the two histidine partners. Analogous to mutational ϕ-analysis, the relative amount of binding energy is quantified as ψ = ∂ΔΔG‡/∂ΔΔGeq. The change in kinetic response due to metal binding reflects the difference between the binding affinity of the site in the TSE relative to the affinity in the unfolded state. A region that is native-like in the TS will realize its full metal binding energy in the TS (ψ=1), while a region that is unstructured in the TS will not (0≤ψ<1). By bridging the side-chains of two residues distant in sequence but structurally local, metal-ion binding serves as a direct probe of long-range interactions and hence, directly reports on chain topology in the TSE. Figure 1 Comparison of metal-ion binding and crosslinking results Another method designed for exploring long-range interactions and chain topology utilizes covalent cross-links. Engineered disulfide bonds created using bicysteine (biCys) substitutions have been utilized in kinetic folding studies (10–12). Using the unmodified biCys variant as the wild-type condition, the change in folding activation energy relative to the change in equilibrium stability due to the formation of the cross-link is calculated as a ϕcrosslink-value, similar to mutational ϕ-analysis. Unlike either mutagenesis or metal-ion binding, cross-linking is believed to act primarily on the unfolded state; stabilizing the protein by reducing the configurational entropy of the unfolded chain. This change in entropy is referred to as “loop closure” entropy (13). The ϕcrosslink-value reflects the degree to which the loop enclosed by the cross-link has lost configurational entropy in the TS. The amount of entropy lost is an indication of the difference between the extents to which the ends of the loop are closed in the TS before and after the introduction of the cross-link. If the loop is unstructured or completely closed in the TS, the change in kinetic response is expected generate a ϕcrosslink-value of zero or one, respectively (Fig. 2A,B). Figure 2 Interpretation of cross-linking and metal ion binding data The interpretations of the results of cross-linking experiments are sometimes at odds with those of the results of mutational ϕ-analysis. In experiments performed in barnase by Fersht and co-workers, the results of the two methods present conflicting pictures of the TS (10). As expected, a crosslink in a region believed to be unstructured in the TS results only in changes to the rate of unfolding. Contrary to expectation, however, a second crosslink in a region believed to be structured causes changes in both the rate of folding and unfolding. The result of the second cross-link suggests that a region believed to be structured in the TS based on mutational ϕ-analysis is not fully structured. Cross-linking experiments by Baker and co-workers on src SH3 also resulted in some ambiguities (11). Of three sites they examined, only the results for one site readily conformed to expectations based on mutational work. The results of cross-linking work in CD2 by Clark and co-workers were similarly mixed (12). It is unclear whether the source of the discrepancies between cross-linking and mutational ϕ-analysis are due to interpretation of the cross-linking or the mutational data. Kiefhaber and co-workers removed two naturally-occurring disulfide bonds in tendamistat and concluded that a local hairpin was formed (14) although no direct comparison with ϕ-values was performed. Here we investigate the suitability of cross-links as probes of long-range interactions in the TS. We also consider the utility of the artificial cross-linker dichloroacetone (DCA) for intra-molecular cross-linking. We find that the cross-linking results for Ub cannot always be interpreted in a manner consistent with both the picture of the TSE presented by ψ-analysis and the prevailing model of protein stabilization by a cross-link. Our results suggest that the sites at which the interpretations of the results of the two methods are consistent are the sites that span topologically simple loops (e.g. a hairpin). Sites at which the results of the two methods cannot be reconciled appear to span more topologically complicated regions. In addition, the apparent discrepancy likely is due to the different manner in which each method stabilizes the protein. Ideally, cross-linking stabilizes the protein by reducing the configurational entropy of the unfolded chain whereas metal-ion binding stabilizes native-like structures where the biHis sites are binding competent. Due to a variety of factors, the effects of cross-linking may not always be restricted to the unfolded chain. When cross-linking affects both the denatured and native states, interpretation becomes difficult. Cross-linking across topologically complicated regions may increase likelihood that the effect will not just be on the unfolded chain. We conclude that cross-linking can be an effective tool for identifying structure in the TS if appropriately placed and interpreted according to the amount of structure formed within the enclosed loop, rather than at the site of the cross-link itself.