Using peptides to study the folding of a predominantly β-sheet protein

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While the primary sequence of a protein is sufficient to guide it to its folded state under native conditions from an ensemble of unfolded conformations in the presence of denaturant, we do not yet know the relative importance of local sequence-directed conformational preferences and global information such as the pattern of hydrophobic and hydrophilic residues. We have described the folding pathway of cellular retinoic acid binding protein I (CRABPI), a predominantly β -sheet protein with an internal cavity that binds retinoic acid, based on results from a variety of methods (stopped flow fluorescence and circular dichroism, quenched flow hydrogen exchange) [1,2]: An early kinetic phase (< 10 msec) appears to correspond to hydrophobic collapse and is accompanied by development of substantial native-like CD signal. The topology of the protein including presence of the central ligand binding cavity develops in a 100 msec kinetic phase [2]. Intriguingly, stable hydrogen bonding in the β sheets is present only after a longer kinetic phase (1 sec), which is also accompanied by the development of native tertiary interactions. A minor population of the molecules folds more slowly (15 to 20 sec) due to slow cis-trans isomerization around X -Pro bonds. We are now examining the importance of local sequence in this folding pathway in two ways: 1) by studies of peptide fragments; and 2) by studies of mutated versions of CRABPI. Thus far, we have found that the small helix–turn–helix of CRABPI has a strong locally driven folding tendency including a sequence motif called a Schellman motif [3]. In the present study, we have focused on the roles that local sequences in turns may play in the folding of CRABPI. The topology of a β -sheet protein requires that non-local segments of chain make specific contacts, yet local sequence must play a role early in folding to favor these interactions. The turns connecting strands are likely to facilitate the formation of the adjacent strand-strand contacts. In CRABPI, like many of its family members, tight turns connect most of the β strands. We have examined characteristics of the tight turns in CRABPI that may point out key local sequences for folding. If a turn is involved in guiding the chain towards the native topology, then one would expect it to be stabilized largely by interactions within its own sequence, even in the context of the native protein