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 proteins that are primarily beta-sheet and contain a large interior cavity into 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 curve 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 stability. All double or triple mutations that include mutation of Leu 64 result in a large and almost identical loss of stability from the wild-type. 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 change in intrinsic fluorescence, this mutant (and similar triple mutants lacking leucine at position 64) folds much more rapidly than wild-type. The mutant, and others that lack Leu 64, have far-UV CD spectra similar to wild-type, but a different near-UV CD spectrum. The folded form of the protein binds oleate, although less tightly than wild-type. Hydrogen/deuterium exchange studies using electrospray mass spectrometry 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 by the D and E strands and that the interaction of Leu 64 with other hydrophobic residues located nearby may be responsible for (1) the slow step in the refolding process and (2) the final stabilization of the structure. We suggest the possibility that this region of the protein may be involved in both an early and late step in refolding.
Kim
K
K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Ramanathan
R
R
Frieden
C
C
D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D050556:Fatty Acid-Binding Proteins; D006860:Hydrogen Bonding; D007422:Intestines / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D016297:Mutagenesis, Site-Directed; D018996:Myelin P2 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins; D019301:Oleic Acid / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D019301:Oleic Acid