Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions

Significance The tools of structural biology afford high-resolution descriptions of folded states of proteins. However, an atomic-level description of unfolded states under folding conditions has remained elusive. Challenges arise from the pronounced conformational heterogeneity and the very low population of unfolded states under folding conditions. We have combined a series of time-resolved biophysical experiments with all-atom simulations and polymer theory to obtain a high-resolution description of unfolded ensembles under folding conditions. These unfolded states are characterized by discernible sequence-specific conformational preferences. These preferences are averaged over by conformational fluctuations, giving rise to ensemble-averaged properties that are consistent with those of canonical random coils. Our findings are relevant to understanding functional and pathological interactions involving unfolded forms of proteins. Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.