| The protein folding problem remains one of the fundamental mysteries of molecular biology. A continuing goal of experimental protein folding is to elucidate the structure of the transition state in the hopes of understanding the forces that drive the process.; First, we examine the utility of covalent crosslinking as a tool for inferring the extent of structure formation in the transition state ensemble. Although crosslinking appears to be prone to artifacts, possibly arising from native-state strain, conditions contributing to the occurrence of such artifacts have been identified. Crosslinking can be another useful tool for studying protein folding; provided it is employed under those circumstances it is most likely to be successful.; Second, we examine the diversity of the transition state ensemble of mammalian ubiquitin using a correlational approach employing multiple simultaneous perturbations. By introducing a crosslink at one site, while, its effect on the Psio-value at a second site is measured, we examined the correlations of sites that are fractionally populated in the transition state ensemble. We found that the TSE populations of the fractional sites examined were independent of each other and were unaffected by perturbations at distal sites. The results of these experiments indicated that a considerable amount of diversity is possible in the TSE of Ub, suggesting a possible entropic role for the fractional sites in stabilizing the TSE in folding. |
