Differences in the folding transition state of ubiquitin indicated by φ and ψ analyses

Abstract

We compare the folding transition state (TS) of ubiquitin previously identified by using analysis to that determined by using analysis. Both methods attempt to identify interactions and their relative populations at the rate-limiting step for folding. The TS ensemble derived from analysis has an extensive native-like chain topology, with a four-stranded ␤-sheet network and a portion of the major helix. According to analysis, however, the TS is much smaller and more polarized, with only a local helix͞ hairpin motif. We find that structured regions can have values far from unity, the canonical value for such sites, because of structural relaxation of the TS. Consequently, these sites may be incorrectly interpreted as contributing little to the structure of the TS. These results stress the need for caution when interpreting and drawing conclusions from analysis alone and highlight the need for more specific tools for examining the structure and energetics of the TS ensemble.multiple pathways ͉ phi analysis ͉ protein folding ͉ transition state M utational analysis has been a major method for characterizing the structure of transition states (TSs) for protein folding (1, 2) and other reactions (3,4). In this approach, the energetic effect of an amino acid substitution on the foldingactivation energy relative to its effect on equilibrium stability, quantified as , is interpreted as the extent to which a mutated residue is involved in the formation of the TS. Values of zero and one are taken to indicate that the influence of the side chain is either absent or fully present, respectively, in the TS.However, in protein folding studies, interpretational issues arise because most values are fractional, generally lying in the range of 0.1-0.5 (5-14). These intermediate values might be due to partial structure formation in the TS or the presence of multiple TS structures. Furthermore, if multiple, alternative TSs exist, a destabilizing mutation will reduce the fraction of states in the ensemble that involve this residue and, thus, generate a lower than expected value (8,14,15). For example, our earlier work with the GCN4 coiled coil protein found low single-site values (16), which turned out to be caused by alternative nucleation positions rather than by the lack of participation by the mutated position (8,11).Even in the case of a single pathway, a mutation can shift the location of the TS either through Hammond or anti-Hammond behavior (15). In general, the effects of an amino acid substitution can depend on an indeterminate combination of local, long-range, native, and even nonnative interactions, and secondary structure preferences. As a consequence, the translation of fractional energetic changes into the language of TS structure is difficult. Some of the problems may be addressable with multiple mutations at a given site (12,15).The analysis methodology overcomes several of these shortcomings by identifying structure and shifts in TS populations upon perturbation (11). In this method, engineered bi-His metal ion bindin...

Cooperative folding near the downhill limit determined with amino acid resolution by hydrogen exchange

“…This application of ϕ analysis using helix promoting/disrupting substitutions at solvent-exposed positions avoids many of the factors that typically confound the interpretation of ϕ values (38) and has proved consistent with ψ analysis (43,44). We also calculated ϕ biHis to further probe the presence of α2 and α4 in the TSE.…”

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

“…Similarly Dobson (Lindorff-Larsen et al 2004 argues that a small network of hydrophobic contacts defines the native topology in the TSE. Further evidence that the requirement for a nucleus, which involves NLIs and directs the formation of the TSE, is a general feature of the folding mechanism was described by many researchers (Fulton et al 1999;Paci et al 2003;Geierhaas et al 2004;Krantz et al 2004;Sosnick et al 2004;Lappalainen et al 2008;Tsong et al 2008;Samatova et al 2009). …”

Identification and characterization of protein folding intermediates

“…In analogy with the F value analysis, the kinetics and equilibria of folding and unfolding may be determined as a function of M 2+ concentration, to extract structural information about folding intermediates and transition states. According to Sosnick and coworkers [50], a potential advantage of Ψ vs F value analysis lies in the more conservative nature of the mutation introduced in the Ψ analysis, the F value analysis always relying in post-mutation conditions for which the importance of an interaction may be reduced per se by the mutation. A surprising result from Ψ analysis was the presence of parallel folding pathways in all reported studies and a major discrepancy between F and Ψ values measured in the same protein [50].…”

“…According to Sosnick and coworkers [50], a potential advantage of Ψ vs F value analysis lies in the more conservative nature of the mutation introduced in the Ψ analysis, the F value analysis always relying in post-mutation conditions for which the importance of an interaction may be reduced per se by the mutation. A surprising result from Ψ analysis was the presence of parallel folding pathways in all reported studies and a major discrepancy between F and Ψ values measured in the same protein [50]. Two independent studies have later explained such discrepancy by showing that Ψ values cannot be analyzed in the same way as other rate-equilibrium free energy relationships due to the involvement of bimolecular reactions [51,52].…”