Figure 1.

Relation between populations and free energies of states. (A) The example free energy landscape has three wells. N is the compact helical native state, U is the expanded unfolded state containing residual helix, and B is the compact misfolded state rich in β-sheet. The sequence of states N-B-U or B-U-N depends on the choice of reaction coordinate. If the N-B barrier is high, there is a preferred path B-U-N. Therefore if only one reaction coordinate is chosen to describe the system, helix content would be better than radius of gyration. (B) The population in U is very small compared to B or N because U lies several RT above B and N. Clearly, this does not mean that U is not involved in the interconversion from B to N. By tuning the solvent condition or mutating the protein sequence in (C), it is possible to bring U to lower free energy so its population will be larger than B.²⁶ The nature of the populated non-native states preceding folding, and the actual paths taken, are sensitive to initial conditions.⁴¹ A dominant pathway is observed because population is exponentially sensitive to small changes of the free energy (Boltzmann factor). Two pathways are rarely going to lie at exactly the same free energy, although it has been observed.^{44, 45}