Phi-value analysis and the nature of protein-folding transition states

Abstract

Phi values are used to map structures of protein-folding transition states from changes in free energies of denaturation (DeltaDeltaG(D-N)) and activation on mutation. A recent reappraisal proposed that Phi values for DeltaDeltaG(D-N) < 1.7 kcal/mol are artifactual. On discarding such derived Phi values from published studies, the authors concluded that there are no high Phi values in diffuse transition states, which are consequently uniformly diffuse with no evidence for nucleation. However, values of DeltaDeltaG(D-N) > 1.7 kcal/mol are often found for large side chains that make dispersed tertiary interactions, especially in hydrophobic cores that are in the process of being formed in the transition state. Conversely, specific local interactions that probe secondary structure tend to have DeltaDeltaG(D-N) approximately 0.5-2 kcal/mol. Discarding Phi values from lower-energy changes discards the crucial information about local interactions and makes transition states appear uniformly diffuse by overemphasizing the dispersed tertiary interactions. The evidence for the 1.7 kcal/mol cutoff was based on mutations that had been deliberately designed to be unsuitable for Phi-value analysis because they are structurally disruptive. We confirm that reliable Phi values can be derived from the recommended mutations in suitable proteins with 0.6 < DeltaDeltaG(D-N) < 1.7 kcal/mol, and there are many reliable high Phi values. Transition states vary from being rather diffuse to being well formed with islands of near-complete secondary structure. We also confirm that the structures of transition-state ensembles can be perturbed by mutations with DeltaDeltaG(D-N) >> 2 kcal/mol and that protein-folding transition states do move on the energy surface on mutation.

Fig. 1.

Schematics of thermodynamic cycles and free-energy profiles.

Fig. 2.

Three-point Leffler plots for the unfolding of barnase. Mutations are as follows: Thr-26 → Ala → Gly, Lys-27 → Ala → Gly, Ser-28 → Ala → Gly, Glu-29 → Ala → Gly, and Gln-31 → Ala → Gly in helix 2.

Fig. 3.

Plot of Φ_U versus ΔΔG_D-N for mutations in helices 1 and 3 of barnase.

Fig. 4.

Three-point Leffler plots for the following mutations of helix 2 of the B-domain of protein A: Gln-27 → Ala → Gly, Arg-28 → Ala → Gly, Asn-29 → Ala → Gly, Gln-33 → Ala → Gly, and Ser-34 → Ala → Gly.

Fig. 5.

Leffler plots of Ala → Gly scanning mutations in the three helices of the B-domain of protein A.

Fig. 6.

Leffler plot for all mutations in the B-domain of protein A. Filled circles are used for the tertiary probes, and open circles are used for secondary structural probes.

Fig. 7.

Leffler plots for single, double, and triple mutations in helix 1 of barnase plus Ala → Gly scanning at position 12 in the mutant Tyr-17 → Gly. The mutants are as follows: Asp-8 → Ala, Asp-12 → Gly, Asp-12 → Ala, Tyr-13 → Ala, Tyr-13 → Ala/Thr-16 → Ser, Tyr-13 → Ala/Tyr-17 → Ala, Tyr-13 → Ala/Thr-16 → Ser/Tyr-17 → Ala, Gln-15 → Ile, Thr-16 → Ala, Thr-16 → Gly, Thr-16 → Ser, Thr-16 → Ser/Tyr-17 → Ala, Thr-16 → Arg, Tyr-17 → Ala, His-18 → Lys, His-18 → Gln, Tyr-17 → Ala, Tyr-17 → Gly, His-18 → Ala, His-18 → Gly, Asp-12 → Ala/Tyr-17 → Gly, and Asp-12 → Gly/Tyr-17 → Gly.

Fig. 8.

Plots of data relating surface exposure on denaturation kinetics of barnase. m_TS-N is the slope of the plot of ΔG_TS-N versus [urea], and m_D-N is the slope of the plot of ΔG_D-N versus [urea]. m_TS-N is the value at 7.25 M urea, measured for unfolding data acquired between 6 and 8.5 M urea, and it is accurate to ±2%. The ratio of m_TS-N/m_D-N is a measure of the relative solvent exposure of the transition state to the denatured state. The value of m_TS-N is a function of just the difference in solvent-accessible surface area of TS and N and does not depend on the properties of the denatured state.