beta-sheet proteins with nearly identical structures have different folding intermediates

The folding mechanisms of two proteins in the family of intracellular lipid binding proteins, ileal lipid binding protein (ILBP) and intestinal fatty acid binding protein (IFABP), were examined. The structures of these all-be ta-proteins are very similar, with 123 of the 127 amino acids of ILBP havin g backbone and Cg conformations nearly identical to those of 123 of the 131 residues of IFABP. Despite this structural similarity, the sequences of th ese proteins have diverged, with 23% sequence identity and an additional 16 % sequence similarity. The folding process was completely reversible, and n o significant concentrations of intermediates were observed by circular dic hroism or fluorescence at equilibrium for either protein. ILBP was less sta ble than IFABP with a midpoint of 2.9 M urea compared to 4.0 M urea for IFA BP. Stopped-flow kinetic studies showed that both the folding and unfolding of these proteins were nor monophasic, suggesting that either multiple pat hs or intermediate states were present during these processes. Proline isom erization is unlikely to be the cause of the multiphasic kinetics. ILBP had an intermediate state with molten globule-like spectral properties, wherea s IFABP had an intermediate stare with little if any secondary structure du ring folding and unfolding. Double-jump experiments showed that these inter mediates appear to be on the folding path for each protein. The folding mec hanisms of these proteins were markedly different, suggesting that the diff erent sequences of these two proteins dictate different paths through the f olding landscape to the same final structure.