Probe‐dependent and nonexponential relaxation kinetics: Unreliable signatures of downhill protein folding

Abstract

The theoretical suggestion that some proteins may encounter no significant free energy barriers during their folding raises an important question: What experimental signature does this downhill folding produce? Several authors have argued that nonexponential (and especially stretched exponential) or probe-dependent kinetics represent useful experimental signatures of a downhill free energy surface. Here we examine more closely the connection between unusual kinetics and downhill energy surfaces. Simulation of diffusive relaxation dynamics on a variety of generically downhill, two-dimensional free energy surfaces shows that these surfaces do not necessarily produce either probe-dependent or significantly nonexponential kinetics. Conversely, we find that two-dimensional surfaces with significant (>3 k(B) T) energy barriers can readily give rise to nonexponential and probe-dependent kinetics. These results show that downhill folding does not constitute a necessary or sufficient condition for nonexponential and/or probe-dependent folding kinetics. One cannot easily prove or disprove that a protein folds downhill simply by studying its relaxation kinetics.