A tale of two turns: Active and passive roles in folding of a β-clam protein
The relationship between local and non-local interactions is important in understanding the folding mechanisms of β-sheet proteins due to the challenges of cooperative structure formation involving non-contiguous β-strands. Cellular retinoic acid binding protein I (CRABP I), a member of the intracellular lipid binding protein (iLBP) family was used as a model to investigate how local interactions in β-turns relate to the cooperative formation of global contacts in CRABP I folding. A review on several peptide and protein engineering studies explored the active and passive roles of β-turns in folding. Evolutionarily conserved networks in the iLBPs were identified through statistical coupling analysis (SCA). The SCA method revealed coupled positions that define a ligand-binding cluster and a hydrophobic cluster, which were used to correlate β-turn interactions in context of their global contacts found in CRABP I. Previous work in our laboratory showed that short peptides corresponding to turns III and IV of CRABP I have propensities for native-like conformations. (Rotondi, K.S. and Gierasch, L.M. Biochemistry, 42, 7976-85, .) Alanine-scanning mutagenesis on each of the turns, in conjunction with equilibrium and kinetic free energies of folding/unfolding measurements of the turn mutants were performed to delineate the contribution of each of the turn residues in the folding mechanism of the intact protein. Turn IV forms early and may actively form a nucleation region for folding; while turn III forms at later stages, passively allowing other regions of the protein to direct folding. These results point to a model in which turn IV contacts form concurrently with the initial hydrophobic collapse and serves as a nucleation point where subsequent native contacts in CRABP I are stabilized by other structural elements such as turn III. The roles of β-turns in folding are defined by their conservation, turn propensity and proximity to topological features of the protein.