Abstract
The 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.