Ubiquitin folds through a highly polarized transition state.

The small alpha/beta protein ubiquitin has been used as a model system for experimental and computational studies on protein folding for many years. Here, we present a comprehensive phi-value analysis and characterize the structure and energetics of the transition state ensemble (TSE). Twenty-seven non-disruptive mutations are made throughout the structure and a range of phi-values from zero to one are observed. The values cluster such that medium and high values and found only in the N-terminal region of the protein, whilst the C-terminal region has consistently low phi-values. In the TSE, the main alpha-helix appears to be fully formed (two phi-values which specifically probe helical structure are one) and the helix is stabilized by packing against the first beta-turn, which is partially structured. In striking comparison, the phi-values in the C-terminal region are all very low, suggesting that this region of the protein is largely unstructured in the TSE. Data are consistent with a nucleation-condensation mechanism in which there is a highly polarized folding nucleus comprising the first beta-hairpin and the alpha-helix. Data presented from the protein engineering study and phi-value analysis are compared with results from other experimental studies and also computational studies.