| We set out to identify general properties of transitions state ensembles (TSEs) to lead to a deeper understanding of the principles that govern protein folding. From our results, we propose a "70% Rule", which states that proteins will fold through TSEs that adopt ∼70% of their respective native state topology, as quantified by the Relative Contact Order (RCO). Due to the high topological threshold, this rule strongly limits the number of possible topologies a protein may adopt in the TSE. This rule was found to be valid for different protein topologies, i.e. protein A (Baxa et al., 2008), Ubiquitin (Ub) (Krantz et al., 2004), and acyl phosphatase (Acp) (Pandit et al., 2006), which span the range of the ln kf - RCO correlation observed by Plaxco et al. (1998). The determination of the TSEs of these proteins was accomplished using &psgr;-analysis and other methods. The discrepancies between the extent of TS structure identified from &psgr;-analysis and mutational ϕ-analysis may be explained through interpretational issues of the ϕ values. Constrained all-atom simulations of unambiguous &psgr;-based TS models for Ub reveal that even residues with experimentally low ϕ-values may be highly buried and making numerous sidechain contacts in the TSE (Baxa et al., 2009). As a result, contact-based definitions for predicting ϕ will not necessarily capture the experimental values since the latter appears to measure structural rigidity rather than structure formation in the TSE. This observation may apply to other proteins, in particular those that have been identified from ϕ-analysis as having a small polarized TSE and appear to violate the "70% Rule". Finally, we utilize a statistical coil library and all-atom simulation to generate an experimentally validated unfolded state ensemble to calculate the loss in backbone entropy during folding. While the loss in conformational diversity is less than previous measurements, the unfolded state is not structurally biased towards the native state. This result suggests that the search for the native state is resolved by preferred pathways wherein tertiary contacts stabilize nascent native secondary structure elements. |
