Summary: Protein folding is a complex diffusive process on a high-dimensional energy surface (1, 2). Gaining detailed insight into the folding energy landscape is an experimental challenge. First, most measurements can observe only one coordinate and provide one-dimensional (1D) projections (Fig. 1 A ). Second, even though a single-molecule experiment can yield folding/unfolding rate constants of a protein, those rates are determined by two distinct properties: barrier height and intramolecular diffusion constant. Recent advances in single-molecule fluorescence resonance energy transfer (FRET) have allowed us to measure the time the protein spends when it transits from the folded to the unfolded conformation (3). Since by definition, the transition state is the most rarely populated state in a protein’s time trajectory, transition path times must be extremely short and are in fact in the range of microseconds (3⇓⇓–6). Since transition path times mostly depend on the intraprotein diffusion constant, they can be used to separate contributions of diffusion constant and barrier height to the measured rate constants. In an elegant study combining transition path measurements with molecular dynamics (MD) simulations, Chung et al. (7) showed that changing pH from 7.5 to 3.2 increases folding rates of the computationally designed protein α3D 15-fold by merely affecting protein diffusion while leaving the transition barrier height unchanged. Fig. 1. ( A ) Schematics of a free energy landscape of protein folding. A 1D projection is highlighted by the black dashed line. A diffusive transition from … [↵][1]1To whom correspondence may be addressed. Email: mrief{at}ph.tum.de. [1]: #xref-corresp-1-1