AbstractThe folding of predominantly β‐sheet proteins is complicated by the presence of a large number of non‐local interactions in their native states, which increase the ruggedness of their folding energy landscapes. However, forming non‐local contacts early in folding or even in the unfolded state can smooth the energy landscape and facilitate productive folding. We report that several sequence regions of a β‐barrel protein, cellular retinoic acid‐binding protein 1 (CRABP1), populate native‐like secondary structure to a significant extent in the denatured state in 8 M urea. In addition, we provide evidence for both local and non‐local interactions in the denatured state of CRABP1. NMR chemical shift perturbations (CSPs) under denaturing conditions upon substitution of single residues by mutation support the presence of several non‐local interactions in topologically key sites, arguing that the denatured state is conformationally restricted and contains topological information for the native fold. Among the most striking non‐local interactions are those between the N‐ and C‐terminal regions, which are involved in closure of the native β‐barrel. In addition, CSPs support the presence of two features in the denatured state: a major hydrophobic cluster involving residues from various parts of the sequence and a native‐like interaction similar to one identified in previous studies as forming early in folding (Budyak et al., Structure 21, 476 [2013]). Taken together, our data support a model in which transient structures involving nonlocal interactions prime early folding interactions in CRABP1, determine its barrel topology, and may protect this predominantly β‐sheet protein against aggregation.