Kinetics are probe-dependent during downhill folding of an engineered lambda6-85 protein.

The Y22W/Q33Y/G46,48A mutant of the protein lambda6-85 folds in a few microseconds at room temperature. We find that its folding kinetics are probe-dependent under a strong bias toward the native state, a new signature for downhill folding. The IR- and fluorescence-detected relaxation time scales converge when the native bias is removed by raising the temperature, recovering activated two-state folding. Langevin dynamics simulations on one- and 2D free energy surfaces tunable from two-state to downhill folding reproduce the difference between the IR and fluorescence experiments, as well as the temperature and viscosity trends. In addition, the 2D surface reproduces the stretched exponential dynamics that we fit to the glucose solution experimental data at short times. Nonexponential dynamics at <10 micros is a signature either for local free energy minima along the reaction coordinate ("longitudinal roughness"), or for folding on a higher-dimensional free energy surface ("transverse roughness").