INTESTINAL FATTY-ACID-BINDING PROTEIN - A SPECIFIC RESIDUE IN ONE TURN APPEARS TO STABILIZE THE NATIVE STRUCTURE AND BE RESPONSIBLE FOR SLOW REFOLDING

The intestinal fatty acid binding protein is one of a class of protein s that are primarily beta-sheet and contain a large interior cavity in to which ligands bind. A highly conserved region of the protein exists between two adjacent antiparallel strands (denoted as D and E in the structure) that are not within hydrogen bonding distance. A series of single, double, and triple mutations have been constructed in the turn between these two strands. In the wild-type protein, this region has the sequence Leu 64/Gly 65/Val 66. Replacing Leu 64 with either Ala or Gly decreases the stability and the midpoint of the denaturation curv e somewhat, whereas mutations at Gly 65 affect the stability slightly, but the protein folds at a rate similar to wild-type and binds oleate . Val 66 appears not to play an important role in maintaining stabilit y. All double or triple mutations that include mutation of Leu 64 resu lt in a large and almost identical loss of stability from the wild-typ e. As an example of the triple mutants, we investigated the properties of the Leu 64 Ser/Gly 65 Ala/Val 66 Asn mutant. As measured by the ch ange in intrinsic fluorescence, this mutant (and similar triple mutant s lacking leucine at position 64) folds much more rapidly than wild-ty pe. The mutant, and others that lack Leu 64, have far-UV CD spectra si milar to wild-type, but a different near-UV CD spectrum. The folded fo rm of the protein binds oleate, although less tightly than wild-type. Hydrogen/deuterium exchange studies using electrospray mass spectromet ry indicate many more rapidly exchangeable amide protons in the Leu 64 Ser/Gly 65 Ala/Val 66 Asn mutant. We propose that there is a loss of defined structure in the region of the protein near the turn defined b y the D and E strands and that the interaction of Leu 64 with other hy drophobic residues located nearby may be responsible for (1) the slow step in the refolding process and (2) the final stabilization of the s tructure. We suggest the possibility that this region of the protein m ay be involved in both an early and late step in refolding.