A "Link-Psi" strategy using crosslinking indicates that the folding transition state of ubiquitin is not very malleable.

Using a combined crosslinking-ψ analysis strategy, we examine whether the structural content of the transition state of ubiquitin can be altered. A synthetic dichloroacetone crosslink is first introduced across two ß strands. Whether the structural content in the transition state ensemble has shifted towards the region containing the crosslink is probed by remeasuring the ψ value at another region (ψ identifies the degree to which an inserted bi-Histidine metal ion binding site is formed in the transition state). For sites around the periphery of the obligate transition state nucleus, we find that the resulting changes in ψ values are near or at our detection limit, thereby indicating that the structural content of the transition state has not measurably changed upon crosslinking. This work demonstrates the utility of the simultaneous application of crosslinking and ψ-analysis for examining potential transition state heterogeneity in globular proteins.

Intramolecular cross-linking evaluated as a structural probe of the protein folding transition state.

We examine the utility of intramolecular covalent cross-linking to identify the structure present in the folding transition state. In mammalian ubiquitin, cysteine residues located across two beta-strands are cross-linked with dichloroacetone. The kinetic effects of these covalent cross-links in ubiquitin, and engineered disulfide bonds in src SH3 (Grantcharova, V. P., Riddle, D. S., and Baker, D. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7084-7089), are compared to the results of psi-analysis where strand association is stabilized by metal ion binding to engineered bihistidine sites (Krantz, B. A., Dothager, R. S., and Sosnick, T. R. (2004) J. Mol. Biol. 337, 463-75) at the same positions. The results for the two methods agree at some of the sites. The cross-linking phi crosslink-values agree with their corresponding psi-values when they have both have values of zero or one, which represent the absence and presence of native structure, respectively. When phi crosslink > psi, the apparent inconsistency is rationalized by the difference between each method's mode of stabilization; cross-linking reduces the configurational entropy of the unfolded state whereas metal binding directly stabilizes the native state. However, when the cross-linking phi-values are smaller than their corresponding psi-values, the apparent underestimation of structure formation is difficult to rationalize while retaining the assumption that the cross-link exclusively affects the entropy of the unfolded state. The interpretation also is problematic for data on cross-links located across strands which are not hairpins, and hence, these sites are likely to be of limited utility in folding studies. We conclude that cross-linking data for sites on hairpins generally report on the amount of structure formed within the enclosed loop while the metal binding data report on the amount structure formed at the site itself.