Biophysical studies of fatty acid binding proteins and hydrophobic ligands.

摘要

Fatty acid binding proteins (FABPs) or lipid binding proteins in general share structural as well as functional characteristics in tissues of different organisms. To determine the molecular basis of the biophysical properties of fatty acid binding proteins, the solution state structure of a muscle fatty acid binding protein isolated from Locusta migratoria was investigated by multi-dimensional NMR. The sequential assignment of ∼85% of the amino acid sequence of this protein was achieved by the use of 2-D homonuclear NMR data. The secondary structure elements, including the β-sheet of the protein, were determined from the NMR data.; Another project attempted to isolate and purify (>90% purity) α ₂-μ-globulin fragment (A2-f), a 15.5 kDa fatty acid binding protein, from kidneys of male rats in order to determine its structure by the use of NMR. α₂-μ-Globulin fragment (A2f) binds fatty acids in vitro and has 100% sequence homology with amino acids 10–159 α ₂-μ-globulin (A2), an 18.6-kDa protein that is synthesized in the male rat liver and is present in the male urine. This purification was unsuccessful but it did reveal heterogeneity of this protein as isolated from tissue.; A third study investigated the mechanism by which sulfonyl urea derivatives, a group of hydrophobic compounds, are transported across a lipid membrane. Sulphonyl ureas are used as drugs for the treatment of type II diabetes mellitus to stimulate the secretion of insulin from the β-cells. The method was to trap a pH-sensitive fluorescent dye to measure pH inside unilamellar vesicles after addition of sulfonyl ureas to the external buffer. It was shown that two sulfonyl ureas, tolbutamide and glybenclamide, diffuse across the bilayer by a “free diffusion” mechanism similar to that of fatty acids. The sulfonyl urea-binding site is located in the cytosolic leaflet of the plasma membrane. Therefore, the drugs can reach receptor binding sites without specialized transport mechanisms.; These studies reveal both properties of hydrophobic molecules in protein-free membranes and structural features of proteins that bind lipids.