Intestinal fatty acid binding protein (IFABP) is a member of the lipid binding protein family, members of which have a clam shell type of motif formed by two five-stranded β-sheets. Understanding the folding mechanism of these proteins has been hindered by the presence of an unresolved burst phase. By initiating the reaction with a sub-millisecond mixer and following its progression by Trp fluorescence, we discovered three distinct phases in the folding reaction of the W6Y mutant of IFABP from which we postulate the following sequence of events. The first phase (k1 > 10 000 s-1) involves collapse of the polypeptide chain around a hydrophobic core. During the second phase (k2 ∼ 1500 s-1), β-strands B-G, mostly located on the top half of the clam shell structure, propagate from this hydrophobic core. It is followed by the final phase (k3 ∼ 5 s-1) involving the formation of the last three β-strands on the bottom half of the clam shell and the establishment of the native hydrogen bonding network throughout the protein molecule.
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